Cell Biology International (2007) 31, 263-268 - Cindy Araya and Bruno Lomonte - Antitumor effects of cationic synthetic peptides derived from Lys49 phospholipase A2 homologues of snake venoms

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Cell Biology International (2007) 31, 263–268 (Printed in Great Britain)

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Antitumor effects of cationic synthetic peptides derived from Lys49 phospholipase A2 homologues of snake venoms

Cindy Araya and Bruno Lomonte^*

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 2060, Costa Rica

Abstract

The effects of two cationic synthetic peptides, derived from the C-terminal region of Lys49 phospholipase A2 homologues from snake venoms, upon various murine tumor cell lines (B16 melanoma, EMT6 mammary carcinoma, S-180 sarcoma, P3X myeloma, tEnd endothelial cells) were evaluated. The peptides are 13-mers derived from Agkistrodon piscivorus piscivorus Lys49 PLA2 (p-AppK: KKYKAYFKLKCKK) and Bothrops asper Lys49 myotoxin II (pEM-2[d]: KKWRWWLKALAKK), respectively, in the latter case with slight modifications and with all-d amino acids. All tumor cells tested were susceptible to the lytic action of the peptides. The susceptibility of tumor cell lines was not higher than that of C2C12 skeletal muscle myoblasts, utilized as a non-transformed cell line control. However, in a murine model of subcutaneous solid tumor growth of EMT6 mammary carcinoma, the intraperitoneal administration of pEM-2[d] caused a tumor mass reduction of 36% (p<0.05), which was of similar magnitude to that achieved by the administration of paclitaxel, an antitumor drug in clinical use. Thus, the C-terminal peptides of Lys49 phospholipase A2 homologues present antitumor effects that might be of interest in developing therapeutic strategies against cancer.

Keywords: Cationic peptides, Antitumor, Phospholipase A2, Snake venom, Myotoxin.

^*Corresponding author. Tel.: +506 229 0344; fax: +506 292 0485.

1 Introduction

Cationic peptides are widely distributed in living organisms, playing a variety of functions. They are often referred to as antibacterial or antimicrobial peptides, due to their well-characterized role in innate immunity against infectious agents (Hancock and Scott, 2000; Boman, 2003). A common property of cationic peptides is their ability to permeabilize biological membranes, an effect which can be selective towards prokaryotes, or can be exerted upon both prokaryotic and eukaryotic cells, depending on the structural features of each peptide (Shin et al., 2000; Glukhov et al., 2005). While most of the studies on cationic peptides have focused on their antimicrobial activity, other biological effects are emerging, and in the past few years the ability of some peptides to affect tumor cells has been reported (Wang et al., 2000; Papo et al., 2003; Okumura et al., 2004; Furlong et al., 2006).

Our laboratory has previously characterized a group of basic phospholipases A2 (PLA2) present in snake venoms, which induce necrosis of skeletal muscle fibers at the site of injection (Gutiérrez and Lomonte, 1997). A naturally-occurring subgroup of these PLA2 myotoxins has been shown to be devoid of enzymatic activity due to critical amino acid substitutions, including the replacement of Asp49 by a lysine, and therefore such variants have been referred to as Lys49 PLA2 homologues (reviewed by Lomonte et al., 2003a). Despite their lack of catalytic activity, these proteins are still able to induce muscle necrosis in vivo and to rapidly lyse a variety of cell types in vitro. The protein region responsible for such toxic effects in Lys49 PLA2 homologues was identified near their C-terminus (Lomonte et al., 1994), and synthetic peptides representing this region can mimic their toxic activities (Núñez et al., 2001). These short (13-mer) peptides derived from snake venom PLA2s present the same general features of the antimicrobial peptides involved in innate immunity, since they include a combination of positively-charged and hydrophobic/aromatic residues, and indeed it was demonstrated that they are able to reproduce the bactericidal activity of the parent toxins (Páramo et al., 1998; Santamaría et al., 2005b).

Based on the recent reports on the antitumor activity of some cationic peptides, the purpose of the present study was to evaluate if synthetic peptides derived from the bioactive C-terminal region of Lys49 PLA2 homologues from snake venoms could have an effect upon tumor cells. Two peptides were selected, representing the region 115–129 of Agkistrodon piscivorus piscivorus Lys49 PLA2 (Núñez et al., 2001) and of Bothrops asper Lys49 myotoxin II (Lomonte et al., 1994), respectively. In the latter case, a modified version of the original myotoxin II peptide, which was optimized for antibacterial activity (Santamaría et al., 2005a), was utilized. Results indicate that tumor cells are susceptible to the lytic action of these short synthetic peptides, and that their growth in vivo can be inhibited by peptide treatment.

2 Materials and methods

2.1 Synthetic peptides

Peptides p-AppK (KKYKAYFKLKCKK) and pEM-2[d] (KKWRWWLKALAKK) were synthesized using F-moc solid-phase strategy on Rink amide resin (Walker, 1994), either automatically by a commercial provider (SynPep Inc.; p-AppK) or manually in our laboratory (pEM-2[d]). In both cases the amino-terminus was free, while the carboxy-terminus was amidated. The final purity of peptides was higher than 95% by analytical RP-HPLC on a Vydac C4 column (250×4.6mm) eluted at 1ml/min with a 0–70% acetonitrile gradient in 0.1% trifluoroacetic acid, and their observed mass spectrometry values matched their expected formula values. The peptides are 13-mers derived from the sequence 115–129 (in the common PLA2 numbering of Renetseder et al., 1985) of two Lys49 PLA2 homologues isolated from the venoms of Agkistrodon piscivorus piscivorus (p-AppK; Núñez et al., 2001) or Bothrops asper (pEM-2[d]; Santamaría et al., 2005b), respectively. In the latter case, slight variations from the original sequence present in B. asper myotoxin II were introduced, previously selected for enhanced bactericidal action (Santamaría et al., 2005b). The variations in pEM-2[d] consist in a triple replacement of Tyr by Trp to increase hydrophobicity, and the substitution of single Cys and Pro residues by Ala, to avoid possible dimerization by intermolecular oxidation, and to facilitate synthesis, respectively. In addition, this peptide was synthesized with all-d-amino acids, to avoid proteolytic degradation during in vivo experiments. It was previously shown that this d-enantiomer (pEM-2[d]) has the same membranolytic potency as its l-counterpart (Santamaría et al., 2005b). Peptides were stored dry at −20°C and dissolved in 0.04M sodium phosphate, 0.12MNaCl, pH 7.2 buffer (PBS) immediately before use.

2.2 Cell lines

The following tumor cell lines of murine origin, obtained from the American Type Culture Collection, were utilized to study the cytolytic effect of synthetic peptides: B16 melanoma (CRL-6323), EMT6 mammary carcinoma (CRL-2775), S-180 sarcoma (TIB-66), and P3X myeloma (CRL-1580). In addition, tEnd cells, a polyoma virus-transformed cell line of murine capillary endothelial origin (Bussolino et al., 1991) was studied. The murine C2C12 skeletal muscle myoblast cell line (CRL-1772) was included as a control of non-transformed cells, known from previous studies to be a suitable target for the cytotoxic action of parent Lys49 PLA2 toxins from which synthetic peptides were derived (Lomonte et al., 1999). Cells were grown under a humidified atmosphere with 7% CO2 at 37°C, in Dulbecco's-modified Eagle medium (DMEM, Sigma) supplemented with 15% fetal calf serum, 2mM glutamine, 1mM pyruvic acid, penicillin (100U/ml), streptomycin (0.1mg/ml), and amphotericin B (0.25μg/ml). Cells were grown routinely in 25cm² bottles, and replicated after dispersion with trypsin (1500U/ml) containing 5mM EDTA, for 5–7min at 37°C. For cytolytic activity determinations, cells were seeded into 96-well plates, at an initial density of approximately 1–4×10⁴cells/well, and allowed to grow for 3–5days, as described.

2.3 Cytolytic activity

After reaching near-confluence in the 96-well plates, cell growth medium was removed by aspiration, and varying amounts of peptides (7.5, 15, 30, 60 and 120μg/well; corresponding to 27.5, 55, 110, 200, and 440μM concentrations, respectively) were immediately added to the cultures, in a total of 150μl of DMEM containing 1% fetal calf serum (Lomonte et al., 1999), in duplicate wells. Cytolysis was determined by measuring lactate dehydrogenase (LDH) activity in cell supernatant aliquots collected after an incubation of 3h with the peptides at 37°C. LDH activity was quantified by a kinetic assay at 340nm (LDH-UV kit, Wiener Laboratories). Control cultures included cells incubated with assay medium alone, or with medium containing 0.1% Triton X-100, in order to establish 0% and 100% reference values for cytolysis.

2.4 Tumor growth inhibition in vivo

EMT6 mammary carcinoma cells (8×10⁵/animal) were injected by subcutaneous route into BALB/c mice (initial body weight of 18–20g). One group of twelve mice was treated with intraperitoneal injections containing 250μg of pEM-2[d] every 48h (corresponding to a dose of 12–13mg/kg of body weight), while a control group (n=12) received similar injections containing only vehicle (PBS) instead. A third group of mice (n=7) was treated intraperitoneally with 90μg (5mg/kg body weight) of the antitumor agent Paclitaxel (Taxol^®, Bristol-Myers Squibb; Wani et al., 1971), every 48h. Mice were observed daily for tumor growth and to record total body weight. Thirteen days after the injection of EMT6 cells all animals were sacrificed by CO2 inhalation, and the solid tumors were excised and weighed. Statistical comparison of differences among the groups was performed by ANOVA, followed by Tukey–Kramer Multiple Comparisons Test, using the GraphPad InStat v.3.07 software.

3 Results

Both synthetic peptides evaluated, p-AppK and pEM-2[d], showed a rapid cytotoxic effect against the different cell lines, as indicated by the release of LDH to the supernatants within 3h (Fig. 1). Cell damage was also evidenced microscopically, by the drastic morphological alterations in the different cell types (Fig. 2). Previous work reported that a scrambled version of p-AppK is completely devoid of cytolytic activity (Lomonte et al., 2003b), ensuring that the lytic action of these cationic peptides, despite being exerted at relatively high concentrations, cannot be attributed to a non-specific effect.

Fig. 1

Cytolytic effect of synthetic peptides upon murine tumor cell lines in vitro. Variable amounts of peptides were added to the cells in a final volume of 150μl, and incubated for 3h at 37°C. Then, the lactate dehydrogenase (LDH) activity of supernatants was determined as an index of cytolysis, as described in Section 2. (○) pAppK, (□) pEM-2[d]. Cell lines correspond to: B16 melanoma (A); S-180 sarcoma (B); P3X myeloma (C); EMT6 mammary tumor (D); tEnd endothelial cells (E); and C2C12 skeletal muscle myoblasts (F). Each point represents mean±SD of duplicate cultures.

Fig. 2

Cytolytic effect of synthetic peptide pEM-2[d] upon EMT6 mammary tumor cells in vitro. (A) Control culture; and (B) peptide-treated (55μM) culture, 3h after incubation.

In general, peptide p-AppK was slightly more potent than pEM-2[d] against the various tumor cell lines, except for the P3X myeloma cells, which where slightly more susceptible to pEM-2[d]. Nevertheless, the differences observed among the various tumor cell types varied within the same order of magnitude, with 50% cytolytic concentration values (IC50) ranging between 50–350μM for both peptides (Table 1). The in vitro cytolytic potency of the synthetic peptides for tumor cell lines was comparable to that observed for the non-transformed C2C12 murine myoblasts (Fig. 1, Table 1), indicating that the peptides do not have a marked selectivity for tumor cells.

Table 1.

Cytolytic concentrations 50% (IC₅₀) of C-terminal-derived synthetic peptides of Lys49 phospholipase A₂ homologues upon murine tumor cell lines in vitro

Peptide	Cell lines
	B16	EMT6	S-180	tEnd	P3X	C2C12*
pEM-2[d]	161 μM	178 μM	317 μM	162 μM	78 μM	184 μM
p-AppK	139 μM	56 μM	172 μM	156 μM	84 μM	139 μM

An in vivo evaluation of the antitumor effect of pEM-2[d], when administered by the intraperitoneal route in mice that had received 8×10⁵ EMT6 cells subcutaneously, resulted in a statistically significant (p>0.05) reduction in tumor mass of approximately 36%, after 13days (Fig. 3). This reduction was of similar magnitude to that caused by the administration of the antitumor drug paclitaxel, at the same time interval (Fig. 3).

Fig. 3

(A) Effect of the intraperitoneal injection of peptide pEM-2[d] on the growth of EMT6 mammary carcinoma cells in BALB/c mice. Tumor cells (8×10⁵) were injected subcutaneously into three groups of mice. The untreated group (n=12) received phosphate-buffered saline alone, whereas experimental groups were treated with either 250μg of pEM-2[d], corresponding to 12mg/kg of body weight (n=12), or 90μg of paclitaxel, corresponding to 5mg/kg of body weight (n=7), every 48h, by intraperitoneal route. The weight of excised tumor masses was determined after 13days. Bars represent mean±SD of each group. (*) indicates a statistically-significant difference (p<0.05) in comparison to untreated controls. (B) Macroscopic comparison of the excised tumor masses in the three groups (n=7).

4 Discussion

The vast majority of studies on cationic peptides have addressed their antibacterial/antimicrobial activities, while only in more recent years other biological properties exerted by these molecules have been reported, including their actions against tumor cells in vitro and in vivo (Wang et al., 2000; Yang et al., 2002; Papo et al., 2003, 2004; Papo and Shai, 2003; Okumura et al., 2004; Furlong et al., 2006). In this study we report that 13-mer synthetic peptides derived from the cationic/hydrophobic C-terminal region of Lys49 PLA2 homologues present in snake venoms, display antitumor effects. A number of studies have reported antitumor activities of individual toxins purified from snake venoms (Braganca and Hospattankar, 1978; Pereira-Bittencourt et al., 1999; Correa et al., 2002; Cura et al., 2002), but there is little information on the use of short segments derived from a snake toxin, in the form of synthetic peptides. Due to their low molecular mass (<1800), it is unlikely that these synthetic peptides would induce an immune response upon repeated administration in vivo. Moreover, their use would overcome the limitations inherent to whole toxin administration, i.e. lethality or other physiopathological effects, observed in some studies (Costa et al., 1997; Cura et al., 2002). In the present work, mice appeared to tolerate peptide injections corresponding to approximately 12mg/kg body weight, with no grossly visible deleterious effects.

Although cationic peptides commonly share membranolytic properties, they can vary widely in terms of selectivity towards membranes of prokaryotes and eukaryotes (Shin et al., 2000; Glukhov et al., 2005). Moreover, some studies have reported a striking differential susceptibility of tumor cells, in comparison to normal cells, to the cytotoxic effects of cationic peptides in vitro, especially when using all-d peptide enantiomers (Papo et al., 2003, 2004; Papo and Shai, 2003). In the present case, no selectivity for the cytolytic effect of the two peptides against transformed and non-transformed cells was observed, even for the all-d enantiomer pEM-2[d]. This would suggest that the d-enantiomeric structure of a cationic peptide, is not a sufficient requirement per se for a high selectivity against tumor cells.

The Lys49 PLA2-derived peptide pEM-2[d] was previously studied in a mouse model of bacterial endotoxemia (Santamaría et al., 2005b). The previous experience with the in vivo use of this peptide prompted to evaluate if its administration could have an effect on the growth of a solid tumor in mice. The EMT6 mammary tumor cells grew rapidly when injected into BALB/c mice and formed well-defined subcutaneous masses in about two weeks, allowing to test the action of pEM-2[d]. Results showed a reduction of about one-third in the weight of EMT6 tumors when animals were treated with 12mg/kg body weight (250μg) of pEM-2[d] every 48h, in comparison to vehicle-treated controls. No differences were observed in total body weight between the animals treated with pEM-2[d] and those receiving vehicle alone. Interestingly, the tumor mass reduction caused by pEM-2[d] was comparable to that obtained with paclitaxel, a clinically approved drug inducing microtubule-mediated mitotic arrest and cell death (Xiao et al., 2006). Taken together, results of the present work indicate that PLA2-derived cationic peptides may have a potential as antitumor agents, which should be characterized in more detail in future studies.

The mode of action of the Lys49 PLA2-derived cationic peptides against tumor cells remains to be investigated. Despite their clear membrane-disruptive effects, here demonstrated at relatively high concentrations in vitro, additional actions such as apoptosis could take place in vivo, at lower concentrations. A Lys49 PLA2 homologue was recently reported to induce apoptosis in vitro, when studied at subcytolytic concentrations (Mora et al., 2005). In addition, it was recently discovered that Lys49 PLA2 homologues from snake venoms interact with the vascular endothelium growth factor (VEGF) receptor-2, with a sub-nanomolar affinity (Yamazaki et al., 2005). This interaction was shown to be functionally relevant in terms of inhibiting the proliferation of endothelial cells stimulated by VEGF165 in culture (Yamazaki et al., 2005). On speculative grounds, such finding would open the possibility that the C-terminal cationic peptides here studied may function as antiangiogenic agents, if they turn out to be involved in the interaction between VEGF receptor-2 and Lys49 PLA2 homologues. Nevertheless, the observation that pEM-2[d] showed an antitumor effect in vivo despite being an all-d stereoisomer, would argue against this possibility. Regardless of the underlying mechanism(s), which remain to be determined, the cationic peptides derived from Lys49 PLA2 homologues present antitumor effects that deserve further exploration.

Acknowledgements

We are grateful to Drs. Marietta Flores, Carlos Santamaría, Yamileth Angulo, and Edgardo Moreno for their valuable collaborations in different aspects of these studies. Financial support from the NeTropica Sweden-Central America Research Network (01-R-2003), and Vicerrectoría de Investigación, University of Costa Rica, is gratefully acknowledged.

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Received 3 August 2006/27 September 2006; accepted 5 November 2006

doi:10.1016/j.cellbi.2006.11.007

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