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INTERNATIONAL JOURNAL OF ONCOLOGY 46: 2181-2193, 2015
CecropinXJ inhibits the proliferation of human gastric cancer
BGC823 cells and induces cell death in vitro and in vivo
YAN-LING WU, LI-JIE XIA, JIN-YAO LI and FU-CHUN ZHANG
Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,
College of Life Science and Technology, Xinjiang University, Tianshan, Urumqi 830046, P.R. China
Received December 19, 2014; Accepted February 5, 2015
DOI: 10.3892/ijo.2015.2933
Abstract. We have shown that an antimicrobial peptide (AMP)
cecropinXJ isolated from the larvae of Bombyx mori selectively inhibits the proliferation of cancer cells. However, the
mechanism remains to be determined. In the present study, we
examined the antitumor activity of cecropinXJ against human
gastric cancer BGC823 cells and explored the mechanism. The
results showed that cecropinXJ inhibited the growth of gastric
cancer BGC823 cells in vitro and in vivo. MTT and colony
formation assays indicated that cecropinXJ suppressed cell
proliferation and reduced colony formation of BGC823 cells
in a dose- and time-dependent manner, but without inhibitory effect on normal gastric epithelia GES-1 cells. S-phase
arrest in BGC823 cells was observed after treatment with
cecropinXJ. Annexin V/PI staining suggested that cecropinXJ
induced both early and late phases of apoptosis through activation of mitochondrial-mediated caspase pathway, upregulation
of Bax expression and downregulation of Bcl-2 expression.
Additionally, cecropinXJ treatment increased reactive oxygen
species (ROS) production, disrupted the mitochondrial
membrane potential (Δψm) and led to release of cytochrome
c. Importantly, in vivo study showed that cecropinXJ
significantly prevented the growth of xenograft tumor in the
BGC823-bearing mice, possibly mediated by the induction of
apoptosis and inhibition of angiogenesis. These results suggest
that cecropinXJ may be a promising therapeutic candidate for
the treatment of gastric cancer.
Correspondence to: Professor Fu-Chun Zhang, Xinjiang Key
Laboratory of Biological Resources and Genetic Engineering, College
of Life Science and Technology, Xinjiang University, 666 Shengli
Road, Tianshan, Urumqi 830046, P.R. China
E-mail: zfcxju@xju.edu.cn
Abbreviations: AMPs, antimicrobial peptides; ROS, reactive oxygen
species; MAPK, mitogen-activated protein kinase; NF-κB, nuclear
factor-kappa B; PI3K/Akt, phosphoinositide 3-kinase/protein kinase B
Key words: cecropinXJ, gastric cancer, antitumor, proliferation,
apoptosis, mitochondrial-mediated pathway, angiogenesis
Introduction
Gastric cancer is one of the most common malignant tumors,
>70% of new cases and deaths occur in developing countries
each year (1). In China, the incidence and mortality rate of
gastric cancer are increasing. Although there are distinct
discrepancies in diagnosis, prognosis, and treatment efficacy
for gastric cancer patients, the 5-year survival rate of gastric
cancer is generally 3.3 mol% corresponding to > 10 wt.%) neutron in-plane
scattering on mechanically aligned membrane systems indicates
the presence of water filled cavities, which resemble ‘wormholes’ [47,59]. The model thus postulates that peptides and lipid
form together well-defined pores [60]. The arguments in favour
or contradicting such a mechanism have been discussed in
considerable detail previously [21,61].
1.2. Carpet model
Based on experimental results obtained with the antibiotic
peptide dermaseptin the carpet model has been proposed
[19,22,62]. This 34-residue antibiotic peptide is rich in lysines
and adopts an amphipathic α-helix (residues 1–27). It thereby
resembles cecropins and magainins for which an in-plane
alignment had been demonstrated [63,64]. The peptide partitions into acidic and zwitterionic membranes [62]. In the
presence of negatively charged lipids and at high peptide
concentrations dermaseptin is located at the membrane surface
and it has been suggested to self-associate in a ‘carpet-like’
manner (Fig. 1B, back). The model further suggests that the
membrane breaks into pieces when a threshold concentration of
peptide is reached [65]. The presence of negatively charged
lipids helps in the formation in a dense peptide carpet, as they
help to reduce the repulsive electrostatic forces between
positively charged peptides.
Whereas the in-plane orientation of these peptides reflects
the detergent-like properties of these amphipathic peptides, the
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carpet model describes the lysis of the membranes at high local
peptide concentrations and reflects the morphological changes
of the membrane after a threshold concentration at the
membrane surface is reached (Fig. 2). Although high peptide
densities have been observed at the surface of bacterial cells, the
concentrations at the membrane itself remain a matter of
speculation [66]. Furthermore, membrane-activities have been
demonstrated at reduced peptide-to-lipid ratios. Indeed comparatively low peptide-to-lipid ratios are needed to dissipate the
ionic gradient across cell membranes or to develop antibiotic
activity [25,37,67]. Notably, it has been speculated that small
openings are sufficient for ion release concomitantly with
inhibition of respiration [25] when at the same time bigger pores
and higher peptide-to-lipid ratios are required for osmolysis of
red blood cells. In a similar manner single-channel recordings of
cecropin or magainin require small amounts of peptide within
the membrane as otherwise lysis occurs [55,56,68]. We
therefore prefer to describe the activities of these peptides by
a more general model which is based on their detergent-like
properties [69–71].
1.3. The ‘detergent-like’ model
Represents the most generally applicable explanation for the
membrane-activities of amphiphiles. The model is based on the
intercalation of these molecules into the bilayer (Fig. 1A).
Thereby, the nature of the molecules such as charge and
hydrophobic volume strongly contribute to the variety of
actions observed and the lipid polymorphism induced [72]. The
interaction of detergents with lipid membranes in particular
above the critical micelle concentration (cmc), where the
molecules exist in their aggregated, micellar state is rather
complex. However, it has to be noted that antimicrobial peptides
can also exist as oligomers (Fig. 1A, top) and thus may exhibit
different behaviour as compared to the monomeric peptides (see
below). Let us consider those general features of detergent–
lipid interactions, which are more related to the action of
antimicrobial peptides. Whereas detergents at very low
detergent-to-lipid ratios can have a neutral effect on model
membranes, or can even result in their stabilization [73,74],
openings might form temporarily at intermediate concentrations, and membrane disintegration becomes apparent at higher
peptide-to-lipid ratios (Figs. 1B and 2). Disintegration of the
bilayer takes into account the loss of the membrane barrier,
dissipation of the transmembrane electrochemical gradient, loss
of cytoplasmic constituents and concomitantly interference with
the energy metabolism of living cells, all being observed also
with antimicrobial peptides. However, it remains possible that,
depending on the peptide and membrane composition, the
peptides merely form small and transient openings (Figs. 1A
and 2) which suffice for the diffusion of smaller molecules,
including the peptides themselves in and out of the cell interior.
The mechanism by which antimicrobial and cytolytic peptides
finally damage the membrane will be determined by the peptide
and lipid structure [42].
The step-wise changes in conductivity that have been
observed for magainin 2 [55–57] or for cecropins [75] are
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Fig. 2. This figure shows a schematic phase diagram of antibiotic peptide/phospholipid mixtures. A variety of macroscopic phases are obtained as a function of peptide/
lipid ratios and lipid composition. Here two lipids with different molecular shapes are mixed and their effect on the macroscopic phase transition in the presence of
antibiotic peptides is shown. For a complete description of antibiotic activities other parameters such as temperature, pH, or cholesterol concentration would have to be
considered.
puzzling observations, which are on first view difficult to
reconcile with an alignment of these peptide helices along the
membrane interface. Interestingly, however, step-wise conductivity increases have also been observed in the presence of
detergents [76,77], in pure lipid membranes [78–80], or when
small unilamellar phospholipid vesicles are added to planar
lipid bilayers [81]. On a molecular level one could thus imagine
that the temporal and local fluctuations of the peptide/detergent
density, related to their lateral and translational diffusion along
interfacial localizations, causes stochastic accumulations of
peptide/detergent in time and space. When high enough local
densities are reached a decrease in ohmic resistance occurs [21].
Alternatively, electroporation of the membrane has been
suggested for NK-lysine an amphipathic helical peptide lying
on the surface of the membrane. In this case the high local
electric fields are sufficient for transient pore formation [82].
An interesting feature of the detergent-like model is the
potential aggregation of the peptides into micelles in aqueous
solution (Fig. 1A, top), whereby an equilibrium between
monomeric peptides and peptide aggregates has to be taken
into account that will result in concentration dependent effects.
For example, tetrameric aggregates have been observed for
melittin under specific conditions [83] and larger aggregates
depending on pH and peptide concentration have been reported
for δ-lysin [84,85]. Self-assembly of peptides was also observed
for natural and synthetic acylated antimicrobial peptides [86–
88]. Such peptide micelles might interact with the lipid bilayer,
e.g. by carrying away lipid molecules which would also result in
transient openings. On the other hand such peptide micelles
could insert (or form) in the lipid bilayer (Fig. 1A), thereby
forming structures that resemble pores without, however, being
well defined in shape or size [89]. The lipopeptaibol trichogin
B. Bechinger, K. Lohner / Biochimica et Biophysica Acta 1758 (2006) 1529–1539
GA IV isolated from the soil fungus Trichoderma longibrachiatum [90] may be an example for such a mechanism.
Leakage and light scattering experiments suggest that within the
explored concentration range membrane perturbation of this
peptide is due to pore formation of the peptide aggregates and
not to micellization [88]. Furthermore, the ability to selfassociate in aqueous medium may be important for target cell
selectivity [86,91]. The role of pre-assembly was tested using
monomeric diastereomeric cationic peptides and their covalently linked pentameric bundles [92]. In contrast to the peptide
aggregates that expressed similar potent antifungal and high
antimicrobial activity as well as haemolytic activity independent on peptide length, the monomeric peptides showed length
dependent antimicrobial activity and were devoid of haemolytic
activity.
A complete description of the peptide–membrane interactions and the resulting membrane morphologies would need to
take into account a wide variety of parameters and conditions.
These include the peptide-to-lipid ratio, the detailed membrane
composition, temperature, hydration and buffer composition.
As with detergents this can be done by establishing phase
diagrams (Fig. 2). Notably, the detergent-like properties of
amphipathic peptides are not in contradiction but include the
above mentioned wormhole and carpet models, in fact these
latter models should be considered ‘special cases’ where the
conditions are such that these kind of supramolecular structures
are observed within a much more complex phase diagram. It
therefore seems tedious to argue about the ‘correct model’ as all
of them might occur depending on the detailed conditions.
Notably, the extensive plasticity of peptide/detergent–lipid
complexes opens up the possibility that a peptide induces a
certain macromolecular structure when interacting with the
membranes of one organism, but a different one when
interacting with another species. On the other hand the
mutagenesis of the peptide sequence causes shifts in the phase
diagram and therefore also affects the biological mechanisms.
In the following we have assembled a number of experimental results which illustrate the detergent-like properties of
amphipathic peptides. When the permeability changes of
membranes are investigated the leakage of fluorescence dyes
takes place at magainin concentrations of approximately 3 mol
% which is equivalent to 81 g/mole lipid. This latter value is
very similar to those observed for the permeability increases in
the presence of the detergents Triton-X100 and octyl glucoside
[76,93].
Furthermore, the macroscopic phase properties as monitored
by proton-decoupled 31P solid-state NMR spectroscopy exhibits interesting parallels when amphipathic peptides, short chain
lipids, lysolipids and detergents are compared to each other
[70,71]. Pore formation due to the increased membrane tension
has also been suggested for other amphipathic helical peptides
[94] and cecropin B activity on bacterial cells was compared to
that of quartenary detergents [95].
When the molecular shape of amphipathic helices and
detergents are compared to each other it becomes evident that,
when intercalated into a lipid bilayer, neither of them fills the
volume at the level of the fatty acyl chains and in the lipid head
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group region equally well (Fig. 3). Whereas the peptide acts as a
spacer at the level of the lipid headgroup it does not occupy the
corresponding volume at the level of the fatty acyl chains
creating a void in the hydrophobic region of the membrane
bilayer [21,42,70]. This parallels the behaviour described for
charged alkyl compounds, which are one of the simplest, among
the variety of detergent molecules being also anchored at the
lipid–water interface with their polar headgroup (for a review
see [72]). Systematic studies using PC model membranes
revealed that molecules with short alkyl chains (C6–12) are
located in the more ordered plateau region of the acyl chain
region of the phospholipids inducing voids below their terminal
methyl groups. Since the formation of such free volume is
energetically unfavourable [96], the hydrocarbon chains must
eliminate these voids by chain bends, increased trans-gauche
isomerization [97] or chain interdigitation as found for very
short chain amphiphiles like alcohols (e.g. [98]) thus affecting
the phospholipids' acyl chain packing. Similar effects were
observed also for non-ionic surfactants such as N-alkyl-N,Ndimethylamine-N-oxides [99] which were shown to exhibit
antimicrobial activity [100,101]. Increasing the size of the polar
headgroup would lead to a molecular shape of the compound
that can be described by a cone which would further promote
the formation of voids.
Amphipathic peptides thus exhibit effects similar to those
observed with cone- or wedge-shaped molecules. Intercalation
of peptides into the membrane interface therefore creates voids
Fig. 3. The molecular shape of lipids and in-plane oriented amphipathic peptides
is illustrated. Whereas lipids such as POPC are considered to exhibit a
cylindrical overall molecular shape, the decreased size of the PE head group
results in an inverted cone structure. On the other hand monoacyl-phospholipids,
detergents or in-plane oriented amphipathic peptides predominantly fill the
space at the head group region/lipid interface thereby resembling a cone. This
model should be considered only as a first order conceptual approach to the real
situation. Notably, the ‘molecular shape’ of the amphipathic molecule reflects
the geometrical as well as the interaction space of these molecules. Therefore
charge and hydrogen interactions between the lipids and the peptide have to be
considered. Furthermore, in-plane oriented peptides can reside as monomers or
as side-by-side oligomers in the membrane (e.g. [125]).
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in the hydrophobic membrane region and in addition imposes
curvature strain on the lipid bilayer [102]. On the other hand the
head group of phosphatidylethanolamine occupies a smaller
area in the interfacial region when compared to the hydrophobic
membrane interior. Therefore, this lipid represents an inverted
cone structure. Due to their shape, mixing cone and invertedcone molecules release the strain and lipid bilayers that exhibit a
tendency to form hexagonal phases e.g. at increased temperatures are stabilized by the insertion of amphipathic peptides
[37,103]. Stabilization of phosphatidylethanolamine bilayers
was also observed for a number of detergents such as Triton X100, deoxycholate or octylglucoside which exhibit a cone
shaped molecular geometry [73].
Considering the large variety of conditions it seems
impossible to establish a complete phase diagram for peptide–
lipid interactions. However, over the years a considerable
amount of data has been collected for melittin. Its overall
amphipathic character composed of a cluster of cationic amino
acids at the C-terminus and a stretch of hydrophobic amino
acids, resembles the features of many detergents being
characterized by a polar/charged headgroup and a hydrophobic
moiety. This peptide is too lytic to be used as an antibiotic but it
can nevertheless illustrate the complexity of such interactions.
There is experimental evidence that melittin inserts into the
membrane interface [104]. The possibility remains that a small
proportion also adopts a transmembrane alignment in the
presence of POPC but not POPG [105,106]. Melittin was found
to exhibit pronounced effects on the phase behaviour of DPPC
already at very low peptide concentrations (lipid-to-peptide
molar ratio of 1000/1) [107,108]. A similar concentration
dependent behaviour was reported for the detergent cetyltrimethylammonium chloride [74]. Since these effects cannot be
accounted for only by local perturbations around the sites of
interaction, long range effects beyond the immediate neighbourhood of the incorporated peptide must be involved.
Therefore, it was suggested that the peptide-affected domains
create line defects in an ordered lipid lattice [104]. Such a
defect-like action at low peptide concentrations may be
explained by shifting the percolation balance of coexisting
gel- and fluid-like states [109]. However, at high melittin
concentrations (lipid-to-peptide molar ratio of 15/1), diskshaped particles were found for the DMPC/melittin mixture
[110,111], suggesting a detergent-like solubilization of the
membrane under these experimental conditions. Below the gel
to fluid phase transition of the pure lipid the presence of
intermediate amounts of melittin (< 5mol%) results in the
reversible disintegration of the bilayer into disks (cf. Fig. 2)
[110,112,113]. It should be noted that some of the mixed
peptide/phospholipid phases exhibit meta-stable properties.
Therefore in the presence of melittin or magainins the
transitions between one phase to another might take many
hours even months [70,114, A. Ramamoorthy, personal
communication].
The idea of gradual membrane disintegration is also
supported by data gained on δ-lysin/PC multilamellar vesicles
[115,116]. This peptide from S. aureus is, like melittin, 26
residues in size and surface-active. It lyses erythrocytes and
many other types of mammalian cells, as well as intracellular
organelles and bacterial protoplasts [117]. Similarly to the
DPPC/melittin system, the addition of small quantities (lipid-topeptide molar ratio ≥ 1000/1) of peptide had only minor but
significant effects on the phase behaviour of DPPC or DMPC
bilayers. Increasing the peptide concentration to molar lipid-topeptide ratios lower than 125/1 promoted the formation of two
populations of lipid particles, which could be separated by
centrifugation. Thereby, small-angle X-ray scattering measurements confirmed that the pellet consists of multilamellar
vesicles, while disk-shaped lipid–peptide aggregates were
found in the supernatant, the predominant aggregate form at
high peptide concentration [116]. Interestingly, the structural
parameters of the DMPC/δ-lysin discoidal aggregates (diameter
of about 14 nm and a bilayer thickness of 5.2nm) correspond
well with the hydrodynamic radius of 6.9 nm estimated for the
disk-shape particles of DMPC/melittin from gel-filtration
experiments [110]. Modelling of the X-ray data suggested a
peptide ring of about 1 nm thickness surrounding the discoidal
lipid bilayer (illustrated in Fig. 2). These data support the idea
that lytic peptides like detergents may have concentrationdependent effects on the membrane structure inducing bilayer
perturbations of long-range order at low peptide concentrations
and exhibiting a detergent-like solubilization of the membrane
at high peptide concentrations. Pore formation, which has also
been reported for δ-lysin [118], is not necessarily in contradiction to this proposal, but may either reflect a transient state or
occur at distinct environmental conditions (Fig. 2). Pore
formation might be particularly sensitive to the lipid class
composing the membrane. Whereas melittin and δ-lysin disrupt
PC bilayers into small structures, the peptides exhibit bilayerstabilizing effects when mixed with phosphatidylethanolamine
membranes, which in the absence of peptide show a tendency
for the inverse hexagonal phase (HII) [119].
A different behaviour is observed when melittin is inserted
into charged phospholipids such as cardiolipin, DOPA or eggPG. In these cases a preference for inverted macroscopic phases
is observed (HII or cubic) [120,121]. Fluorescence spectroscopy
indicates that the macroscopic phase properties depend not only
on the molecular shape of the lipid but also on the insertion
depth of the peptide [122]. Interestingly, the presence of
negative surface charge densities stabilizes PC lipid bilayers in
the presence of melittin [106,111,123,124]. Thus the positioning and topology of melittin, and concomitantly its effect on
membrane structure, are all strongly dependent on a wide
variety of parameters that determine the local environment.
Much less data are available for the peptide–lipid interactions
involving antibiotic peptides. However, comparable correlations
between the molecular shape of the phospholipids and
macroscopic phase transitions have been observed for magainin
or its analogue MSI-78 [70,71]. The peptides have been shown
to be oriented parallel to the membrane surface using solid-state
NMR spectroscopy [64] and MSI-78 to form antiparallel dimers,
when solubilized in dodecylphosphocholine (DPC) micelles
[125]. The latter observation suggests that magainin activity may
be also modulated by oligomerization. Fluorescence energy
transfer measurements indicate that the hydrophobic regions of
B. Bechinger, K. Lohner / Biochimica et Biophysica Acta 1758 (2006) 1529–1539
magainin 2 are located approximately 10 Å from the bilayer
center [126]. The peptide or its analogue MSI-78 thus acts like a
wedge, which augments the curvature of the membrane. By
inserting in-plane peptides into the headgroup region the bilayer
packing is disturbed at an estimated radius around the peptide of
approximately 50 Å resulting in a reduction in the average
bilayer thickness [127–130]. A current atomic force microscopy
study on DMPC bilayers and the magainin analogue MSI-78
also revealed that the membrane thickness is not reduced
uniformly over the entire bilayer area [130]. Deuterium or 13C
solid-state NMR measurements indicate a decrease in order
parameter at the lipid bilayer interior for both magainin and MSI78 [131,132]. More recent investigations indicate that in mixed
PC/PS lipid membranes cationic amphipathic peptides preferentially interact with the acidic phosphatidylserine [133,134]. A
preferential interaction with the negatively charged lipid
component in mixed model membranes was also observed
earlier for human neutrophil peptide, HNP-2 [135], PGLa [53] or
nisin [136] resulting in charge segregation. Such a behaviour can
be expected because of the cationic nature of the peptides, and is
typical for a large number of antimicrobial peptides, such as e.g.
the α-helical peptides belonging to the magainin-family
[35,137] and buforin II [138], the β-sheet peptides tachyplesin
[139] and protegrin-1 [54,140], the cyclic peptides gramicidin S
[141] and rhesus theta defensin, RTD-1 [142] as well as nisin Z
[143]. There is also evidence that other biologically active
amphipathic molecules, such as cardiotoxin [144] and synthetic
peptides [145] interact with model membranes inducing lateral
separation of phospholipids into co-existing domains.
Additional support for the detergent-like model arises from
the observation that antibiotic or model peptides too short to
span the membrane exhibit channel-like activities as well [146–
153]. In particular the model is in perfect agreement with the
experimentally observed in-plane orientation of amphipathic
peptides [45,64,126].
The membrane lipid composition can thus have pronounced
effects on the membrane-disrupting activities of amphipathic
peptides. This is reflected by shifts in the borders within the
peptide–membrane phase diagram or by the occurrence of
different macroscopic phases when one diagram is compared to
another. The lipid composition thereby modulates the sensitivity
of the membrane to a given peptide [42,53,54,154]. For
example, the alignment and the dynamics of the C-terminal
helical domain of pardaxin are a function of the phosphatidylcholine fatty acyl chain composition [51,52]. Furthermore,
cholesterol which is only present in eukaryotic cells changes the
interactions of cationic peptides with the membrane [68,155].
Cholesterol affects the fluidity and the dipole potential of
phospholipid membranes. Furthermore, its capacity to form
hydrogen bonds with the peptide has also been suggested to
reduce antibiotic activity [156]. However, recent experiments
on δ-lysin showed that in ternary lipid systems mimicking
mammalian cell membranes the peptide binds preferentially to
the liquid-disordered domain suggesting that there is no specific
interaction between δ-lysin and cholesterol and sphingomyelin,
respectively, emphasizing the importance of membrane properties in lipid–peptide interactions [157]. As a result of peptide
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accumulation peptide aggregates may form that become
orientationally ordered, which in turn may have an impact on
several processes such as peptide translocation across membranes [158]. Moreover, peptide enriched lipid domains will
form especially in the presence of anionic lipids that differ
significantly in their local properties from the membrane bulk
phase [42]. This may result in packing defects giving rise to
increased membrane permeability. In addition, exclusion of
certain lipids, e.g. segregation of anionic lipids, from areas of
the cell membrane due to their preferential interaction with the
cationic peptides may affect membrane structure and integrity
[133,134]. Thus, it is evident from the discussion above that the
molecular mechanism of membrane permeation and disruption
obviously depends on a number of parameters such as the nature
of the peptides and membrane lipids, peptide concentration and
environmental conditions.
Acknowledgements
We are grateful to the members of our laboratories for their
important contributions to this subject as well as to many friends
and colleagues for ongoing discussions on this issue. We are
indebted to Sabine Danner for her kind and competent help
during the preparation of the manuscript.
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