Cell Biology International 31 (2007) 263e268

www.elsevier.com/locate/cellbi

Antitumor effects of cationic synthetic peptides derived from

Lys49 phospholipase A2 homologues of snake venoms
Cindy Araya, Bruno Lomonte*
Instituto Clodomiro Picado, Facultad de Microbiologı́a, Universidad de Costa Rica, San José 2060, Costa Rica
Received 3 August 2006; revised 27 September 2006; accepted 5 November 2006

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.
Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Cationic peptides; Antitumor; Phospholipase A2; Snake venom; Myotoxin

1. Introduction
Cationic peptides are widely distributed in living organ-
isms, 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 to-
wards 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,
* Corresponding author. Tel.: þ506 229 0344; fax: þ506 292 0485.
E-mail address: blomonte@cariari.ucr.ac.cr (B. Lomonte).
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 substi-
tutions, 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

1065-6995/$ - see front matter Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.cellbi.2006.11.007
264 C. Araya, B. Lomonte / Cell Biology International 31 (2007) 263e268
(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 demon-
strated that they are able to reproduce the bactericidal activity
of the parent toxins (Páramo et al., 1998; Santamarı́a 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 115e129 of Agkistro-
don 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 (Santamarı́a 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] (KKWRWWL
KALAKK) 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  4.6 mm) eluted at 1 ml/min with a 0e70% 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 115e129 (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]; Santamarı́a 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 (Santamarı́a
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 (Santamarı́a
et al., 2005b). Peptides were stored dry at 20  C and dissolved in 0.04 M
sodium phosphate, 0.12 M NaCl, pH 7.2 buffer (PBS) immediately before use.
2.2. Cell lines
The following tumor cell lines of murine origin, obtained from the Amer-
ican Type Culture Collection, were utilized to study the cytolytic effect of syn-
thetic peptides: B16 melanoma (CRL-6323), EMT6 mammary carcinoma
(CRL-2775), S-180 sarcoma (TIB-66), and P3X myeloma (CRL-1580). In ad-
dition, 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 humidi-
fied atmosphere with 7% CO2 at 37  C, in Dulbecco’s-modified Eagle medium
(DMEM, Sigma) supplemented with 15% fetal calf serum, 2 mM glutamine,
1 mM pyruvic acid, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and
amphotericin B (0.25 mg/ml). Cells were grown routinely in 25 cm2 bottles,
and replicated after dispersion with trypsin (1500 U/ml) containing 5 mM
EDTA, for 5e7 min at 37  C. For cytolytic activity determinations,
cells were seeded into 96-well plates, at an initial density of approximately
1e4104 cells/well, and allowed to grow for 3e5 days, 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 120 mg/well; corresponding to 27.5, 55, 110, 200, and 440 mM concentra-
tions, respectively) were immediately added to the cultures, in a total of 150 ml
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 3 h with
the peptides at 37  C. LDH activity was quantified by a kinetic assay at
340 nm (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 sub-
cutaneous route into BALB/c mice (initial body weight of 18e20 g). One
group of twelve mice was treated with intraperitoneal injections containing
250 mg of pEM-2[D] every 48 h (corresponding to a dose of 12e13 mg/kg
of body weight), while a control group (n ¼ 12) received similar injections
containing only vehicle (PBS) instead. A third group of mice (n ¼ 7) was
treated intraperitoneally with 90 mg (5 mg/kg body weight) of the antitumor
agent Paclitaxel (TaxolÒ, Bristol-Myers Squibb; Wani et al., 1971), every
48 h. 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 TukeyeKramer 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
3 h (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 concentra-
tions, cannot be attributed to a non-specific effect.
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 50e350 mM 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
 C. Araya, B. Lomonte / Cell Biology International 31 (2007) 263e268 265

100 A B

80

60

40

20

100 C D

80
Cytolysis (%)

60

40

20

100 E F

80

60

40

20

0
0 100 200 300 400 500 0 100 200 300 400 500
Peptide ( M)

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 150 ml,
and incubated for 3 h at 37  C. Then, the lactate dehydrogenase (LDH) activity of supernatants was determined as an index of cytolysis, as described in Section 2.
(B) 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.

non-transformed C2C12 murine myoblasts (Fig. 1, Table 1),
indicating that the peptides do not have a marked selectivity
for tumor cells.
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 13 days (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).
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 homo-
logues present in snake venoms, display antitumor effects.
A number of studies have reported antitumor activities of indi-
vidual toxins purified from snake venoms (Braganca and Hos-
pattankar, 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
266 C. Araya, B. Lomonte / Cell Biology International 31 (2007) 263e268

cells, in comparison to normal cells, to the cytotoxic effects

Fig. 2. Cytolytic effect of synthetic peptide pEM-2[D] upon EMT6 mammary
tumor cells in vitro. (A) Control culture; and (B) peptide-treated (55 mM)
culture, 3 h after incubation.
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 ap-
proximately 12 mg/kg body weight, with no grossly visible
deleterious effects.
Although cationic peptides commonly share membrano-
lytic 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
of cationic peptides in vitro, especially when using all-D pep-
tide 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 cat-
ionic 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 (Santama-
rı́a 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
12 mg/kg body weight (250 mg) of pEM-2[D] every 48 h, 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. Interest-
ingly, the tumor mass reduction caused by pEM-2[D] was com-
parable 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. De-
spite their clear membrane-disruptive effects, here demon-
strated 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 re-
ported to induce apoptosis in vitro, when studied at subcyto-
lytic 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 rel-
evant 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

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 mM 178 mM 317 mM 162 mM 78 mM 184 mM
p-AppK 139 mM 56 mM 172 mM 156 mM 84 mM 139 mM
Cytolysis was determined by measuring lactate dehydrogenase (LDH) release to cell supernatants, 3 h after exposure to variable concentrations of synthetic
peptides, as described in Section 2. Values represent means of duplicate assays. *: included as a non-transformed cell line control.
 C. Araya, B. Lomonte / Cell Biology International 31 (2007) 263e268
70
A
60
50
Tumor Weight (mg)
* 1978;14:707e12.
40
30
20
10
0 study of VRCTC-310, a purified phospholipase A2 from snake venom, in
Control pEM-2[D] Paclitaxel
B Phase I and pharmacokinetics study of crotoxin (cytotoxic PLA2, NSC-
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 ¼ 12) received phosphate-buffered saline alone, whereas
experimental groups were treated with either 250 mg of pEM-2[D], correspond-
ing to 12 mg/kg of body weight (n ¼ 12), or 90 mg of paclitaxel, corresponding
to 5 mg/kg of body weight (n ¼ 7), every 48 h, by intraperitoneal route. The
weight of excised tumor masses was determined after 13 days. 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 ¼ 7).
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. Re-
gardless 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 Santamarı́a,
Yamileth Angulo, and Edgardo Moreno for their valuable
collaborations in different aspects of these studies. Financial
support from the NeTropica Sweden-Central America Re-
search Network (01-R-2003), and Vicerrectorı́a de Investiga-
ción, University of Costa Rica, is gratefully acknowledged.
267
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