European Journal of Medicinal Chemistry 89 (2015) 540e548

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Design, synthesis and biological evaluation of novel peptides with

anti-cancer and drug resistance-reversing activities
Xin Deng, Qianqian Qiu, Baowei Yang, Xuekun Wang, Wenlong Huang*, Hai Qian*
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 7 September 2014
Received in revised form
24 October 2014
Accepted 25 October 2014
Available online 28 October 2014

Chemotherapy is an important approach used to treat cancer, but severe side effects and emerging drug
resistance restrict its clinical application. In this present study, we found that peptide B1 showed specic
cytotoxicity to tumor cells. Moreover, a helix-wheel plot predicted that the Ser14 in this peptide is
located at the interface of the hydrophobic and hydrophilic faces of B1. Subsequently, we wondered
whether replacing Ser14 would alter the activity of B1, and so a series of B1 analogs were synthesized
where the Ser14 was replaced by amino acids with distinct physicochemical properties. Amongst them,
peptides where Ser14 was substituted by a nonpolar and basic amino acid had improved anti-cancer
activity. Further investigations revealed that B1 and its analogs were capable of penetrating into cytoplasm and triggering cytochrome C release from mitochondria, which ultimately resulted in apoptosis.
Meanwhile, B1 and its analogs inhibited the migration of cancer cells. The peptides also acted against
drug-resistant cells and had drug resistance-reversing effects. In conclusion, these peptides might be
promising candidates for oncotherapy.
2014 Elsevier Masson SAS. All rights reserved.

Keywords:
Anti-cancer
Drug resistance-reversing effects
Membrane-disruption
Peptides

1. Introduction
Cancer comprises a group of complicated diseases characterized
by the unregulated growth of abnormal cells that have the capability of invading normal tissues, metastasizing to other organs and
spreading to other body parts [1e3]. As a leading cause of death and
disability, cancer is responsible for nearly 7.6 million deaths per
year [4]. Chemotherapy is one of the important approaches used in
cancer therapy, but severe side effects resulting from toxicity of
chemotherapeutics on normal cells remains an important obstacle
in clinical application [5e7]. Meanwhile, another problem
encountered during cancer therapy is drug resistance resulting
from changes in drug transporters or detoxifying enzymes of the
cancer cells [2].

Abbreviations: 5-Fu, 5-uorouracil; ADM, adriamycin; AMPs, antimicrobial

peptides; AO, acridine orange; DMSO, dimethyl sulfoxide; EB, ethidium bromide;
FBS, fetal bovine serum; FITC, uorescein isothiocyanate; JC-1, 5,50 ,6,60 -tetrachlorol,l0 ,3,30 -tetraethyl-benzimidazol-carbocyanine iodide; MTT, thiazolyl blue; RBCs, red
blood cells; RP-HPLC, reversed phase high-performance liquid chromatography;
SEM, scanning electron microscope; UPLC, ultra-high performance liquid chromatography; Ver, Verapamil.
* Corresponding authors.
E-mail addresses: ydhuangwenlong@126.com (W. Huang), qianhai24@163.com
(H. Qian).
http://dx.doi.org/10.1016/j.ejmech.2014.10.072
0223-5234/ 2014 Elsevier Masson SAS. All rights reserved.

Antimicrobial peptides (AMPs) are ancient host defense compounds that have activity against a broad spectrum of microorganisms [8,9]. In general, AMPs possess 12e50 amino acids and, of
these, 2e9 are typically positively charged while about 50% are
hydrophobic amino acids. These residues are spatially organized to
make AMPs amphipathic [10,11]. Recent studies have shown that
several AMPs can exert anti-cancer effects [12].
As a novel type of promising chemotherapeutic, AMPs present
signicant advantages over traditional antitumor agents, such as
higher specicity and circumvention of drug resistance [13,14].
There are essential differences between the cell membranes of
neoplastic and normal cells and these differences might account for
the selective cytotoxicity of AMPs to cancer cells while sparing
healthy cells. Most cancer cells carry a net negative charge on the
membrane due to the overexpression of various anionic molecules,
such as phosphatidylserine and O-glycosylated mucins [15e18]. On
the contrary, healthy eukaryotic cells possess high contents of
zwitterionic membrane components, including phosphatidylethanolamine, phosphatidylcholine and sphingomyelin, which confer
an overall neutral charge on the cell surface [19]. As a consequence,
an electrostatic interaction would occur between the AMPs and
cancer cells rather than with untransformed counterparts.
Several presumptions have been proposed to explain the interactions between AMPs and cell membranes. In brief, the
positively-charged peptides initially bind to and cover the cell

X. Deng et al. / European Journal of Medicinal Chemistry 89 (2015) 540e548

membrane via electrostatic attraction. Then AMPs insert into

membrane when a critical concentration is reached. At this point,
two mechanisms have been proposed: (i) a membrane disruptive
mechanism where the AMPs reorientate and disrupt membrane
integrity resulting in leakage of metabolites and ions as well as
depolarization; (ii) a non-membrane mechanism where the AMPs
translocate into the cytoplasm by virtue of cell membrane perturbation or pore formation so they are free to act on intracellular
targets and cause apoptosis [12,20].
Cathelicidin-BF15 (BF-15) is a 15-mer AMP with broad antimicrobial activity, but its C-terminal amidated derivative B1 (primary
sequence: VKRFKKFFRKLKKSV-NH2) has even greater antimicrobial
potency [21]. The anti-cancer properties of B1 have not been reported previously. In this present study, we demonstrate the anticancer effects of B1 on the MCF-7 breast cancer and K562 leukemia cell lines. Additionally, we show that B1 has relatively low
cytotoxicity against the normal cell line GES-1.
Two common traits, hydrophobicity and positive charge, play
pivotal roles in the activity of AMPs [22,23]. According to a helixwheel plot (http://heliquest.ipmc.cnrs.fr/), we found that the
serine residue at position 14 (Ser14), an uncharged polar amino
acid, is located between the hydrophobic and cationic surfaces of
B1 (Fig. 1). Therefore, we wondered whether replacement of Ser14
by distinct amino acids would alter the anti-cancer activity of B1.
Alanine substitution is a common approach when verifying the
active site of peptides [24], and so we synthesized B1-Ala14
(VKRFKKFFRKLKKAV-NH2) where Ser14 was replaced by alanine
and assessed the antitumor activity. B1-Ala14 exhibited increased
anti-cancer activity compared to B1. Subsequently, we synthesized
11 other peptides and assessed their antitumor activities using the
MTT assay. Peptides where the Ser14 was replaced by a nonpolar or
basic amino acid exhibited more favorable activity against some
cancer cell lines, while all the peptides had lower toxicity against
benign cells.
In order to clarify the anti-cancer mechanism, B1 and two of its
analogs (B1-Leu and B1-Lys) were chosen for further investigation.
Meanwhile, anti-cancer activity was also assessed against adriamycin (ADM)-resistant cell lines.
2. Materials and methods
2.1. Peptide synthesis, purication and analysis

K562/ADM, and the GES-1 gastric epithelial cell line were used in
this study. All the cell lines were obtained from KeyGEN BioTECH
(Nanjing, China). Cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (Hyclone Laboratories) and 1% penicillinestreptomycin antibiotic mixture (Gibco
BRL). All the cell lines were cultured at 37  C in a humidied atmosphere at 5% CO2 and 95% air.
2.4. Cell proliferation and viability assay
Anti-cancer activity of B1 and its analogs was assessed by the
MTT assay. Cells in the logarithmic phase of growth were collected
and seeded at 5  103 cells/well in 96-well plates 24-h before
peptide treatment. The cells were treated with various concentrations of peptides while controls were not exposed to peptide. After
incubating for 48 h, 20 mL of 5 mg/mL MTT solution was added to
each well and allowed to incubate for 4 h. Then, 150 mL DMSO was
added to dissolve the MTT formazan precipitate. The absorbance at
490 nm of the mixture in each well was measured with a Bio-Rad
microtiter plate reader. Average cell survival rate was calculated
by
the
following
formula:
cell
survival
rate
(%) (A0 490nm  A490nm)/A0 490nm  100%, where A0 490nm is absorbance at 490 nm of the sample without peptide and A490nm is
absorbance at 490 nm of the sample with peptide. Survival rates
were plotted against peptide concentrations and the IC50 value for
each cell line was calculated based on the survival curve.
2.5. Hemolysis assay
Rabbit red blood cells (RBCs) were used for hemolysis assay
follow the process described by Chen et al. [21]. In brief, 100 mL of
peptide solution was added to 100 mL of RBCs PBS suspension in 96well plates, which were incubated for 1 h at 37  C and centrifuged
at 1000 g for 5 min. Supernatant were transferred to fresh 96-well
plates and hemoglobin release was measured by microplate reader
(Bio-Rad, iMark 680) at 540 nm. Zero and 100% hemolysis were
determined in PBS and 0.1% Triton X-100, respectively.
2.6. Acridine orange/ethidium bromide (AO/EB) double staining
The MCF-7 cell line was used for AO/EB double staining. Briey,
MCF-7 cells were grown in a 24-well microtiter plate at 1  105 cells

The synthesis of B1 and its analogs was accomplished by solidphase methods on a microwave synthesizer (CEM, NC, USA), and
the general procedure has been reported previously [25]. Crude
peptides were puried by preparative reversed-phase high-performance liquid chromatography (RP-HPLC; Shimadzu LC-10) using
a C18 column (5 mm, 340  28 mm). Purity analysis and characterization was performed by ultra-high performance liquid chromatography/mass spectrometry (UPLC/MS; Waters UPLC with the
ACQUITY TQD; Waters Corporation, Milford, MA, USA) on a Waters
ACQUITY UPLC BEH C18 column (1.7  50 mm, Waters). The purity
of the peptides was above 95%.
2.2. Helix-wheel plot
A helix-wheel plot was achieved using the software package
provided by The Expert Protein Analysis System (ExPASy) proteomics
server as described elsewhere (http://heliquest.ipmc.cnrs.fr/) [26].
2.3. Cell cultures
The MCF-7 breast cancer cell line, an ADM-resistant sub-line
MCF-7/ADM, leukemia K562 cell line, an ADM-resistant sub-line

541

Fig. 1. The helix-wheel plot of B1.

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X. Deng et al. / European Journal of Medicinal Chemistry 89 (2015) 540e548

per well and incubated for 24 h. Then the cells were treated with
20 mM B1, 5 mM B1-Leu or 8 mM B1-Lys for 30 min. The medium was
removed and the cells were washed twice with phosphate-buffered
saline (PBS). The cells were stained in the dark for 10 min with 1 mL
cold PBS containing 40 mL of 1 mg/mL AO and 1 mg/mL EB. After
staining, excess AO/EB was washed off with cold PBS. Fluorescence
imaging was achieved by uorescence microscopy (OLYMPUS
America, Melville, NY).
2.7. Scanning electron microscopy (SEM)
MCF-7 cells were seeded at 1  105 per well into a 6-well microtiter plate containing sterilized coverslips. The plate was incubated for 24 h and then 20 mM B1, 5 mM B1-Leu or 8 mM B1-Lys was
added for 30 min. The medium was removed and the cells were
gently washed with PBS. One milliliter of 2.5% glutaraldehyde solution was added to each well to x the cells. After xation, the
samples were sent to the College of Life Science at Nanjing Agricultural University for further treatment and analysis on a Model S3000N SEM (Hitachi High-Technologies Corporation).
2.8. Peptide localization by uorescein isothiocyanate (FITC)
labeling
The method for synthesizing FITC-labeled peptides has been
reported previously [27]. MCF-7 cells were placed in a 24-well
microtiter plate at 1  105 cells per well and incubated for 24 h.
Then cells were treated with FITC-labeled B1, B1-Leu or B1-Lys.
After incubation for 5 min or 30 min, cells were washed three times
with cold PBS. The accumulation of the FITC-labeled peptides in the
cells was observed by uorescence microscopy (OLYMPUS America,
Melville, NY).
2.9. Assessment of mitochondrial membrane potential (DJ) by
5,50 ,6,60 -tetrachloro-l,l0 ,3,30 -tetraethylbenzimidazolcarbocyanine
iodide (JC-1) assay
MCF-7 cells were seeded at 1  105 per well in a 24-well microtiter plate and incubated for 24 h. Then the cells were treated
with 20 mM B1, 5 mM B1-Leu or 8 mM B1-Lys for 2 h. The variation of
mitochondrial membrane potential (DJ) of MCF-7 cells was
assessed by a Mitochondrial Membrane Potential Assay Kit (Nanjing Jiancheng Bioengineering Institute).
2.10. Assessment of cytochrome C contents
MCF-7 cells were seeded at 2  105 per well in a 6-well microtiter plate and incubated for 24 h. Then the cells were treated
with 20 mM B1, 10 mM B1, 5 mM B1, 5 mM B1-Leu or 8 mM B1-Lys for
2 h. The total protein, mitochondrial protein and cytoplasmic protein were collected with the aid of a Mitochondria Protein Extraction Kit (Nanjing Jiancheng Bioengineering Institute). The
cytochrome C contents in each fraction were evaluated by a Human
Cytochrome C Elisa Kit (Nanjing Jiancheng Bioengineering
Institute).
2.11. Wound healing assay
Cells were seeded in six-well plates at 2  105 cells per well and
incubated for 24 h. After the cells had grown to 90% conuence, a
cell-free gap was created and the cells were allowed to migrate in
serum-free medium for 48 h. Wound closure or cell migration
images were photographed after treatment with 5 mM B1, 0.5 mM
B1-Leu or 1 mM B1-Lys at 0 h and 48 h.

2.12. Transwell migration assay

This assay was employed to study the migratory abilities of tumor cells exposed to the peptides. The upper and lower chambers
of the Transwell (Costar Corp.) plates are separated by Millipore
membranes (pore size of 8 mm). The process of cells moving from
the upper to lower surface of the membrane is considered as
migratory activity. MCF-7 cells (4  104) were added to each upper
chamber with medium containing 5 mM B1, 0.5 mM B1-Leu or 1 mM
B1-Lys. After incubation for 48 h, the migrating cells on the lower
surface were counted.
2.13. Drug resistance-reversing assay
ADM-resistant MCF-7/ADM or K562/ADM cells were seeded in
96-well plates at 1  104 cells per well and incubated for 24 h. Then
5 mM B1, 0.5 mM B1-Leu or 1 mM B1-Lys was added to the wells
followed by various concentrations of ADM. The exponentiallygrowing cells were incubated for 48 h. Then MTT was added to
each well and the plate was incubated for 4 h at 37  C, before 150 mL
DMSO was added and the absorbance at 570 nm was read on a BioRad microtiter plate reader.
2.14. Accumulation of ADM
ADM accumulation was determined according to a reported
procedure with minor modications [28]. Briey, 2  105 cells of
MCF-7/ADM or K562/ADM were incubated with 20 mM ADM and
5 mM B1, 0.5 mM B1-Leu or 1 mM B1-Lys for 2.5 h. Verapamil was
used as the positive control. After incubation, the cells were washed
with cold PBS and observed with uorescence microscopy
(OLYMPUS America, Melville, NY). In addition, cells were also lysed
with lysis buffer (0.75 M HCl, 0.2% Triton-X100 in isopropanol). The
uorescence level of ADM in the lysate was determined by uorescence spectrophotometry (RF-5301 PC; Shimadzu Scientic Instruments Inc., Kyoto, Japan) using excitation and emission
wavelengths of 460 and 610 nm, respectively.
3. Results
3.1. Structural characteristics of B1 and its analogs
The peptide sequences and structural characteristics of B1 and
its analogs are presented in Table 1. After purication of the peptides by preparative HPLC, UPLC/MS was employed to analyze the
purity and molecular weight of the peptides. The mass-to-charge
ratio (m/z) was found to be nearly identical to the theoretical m/z
in each case (as shown in Table 1).
3.2. Anti-cancer activity of B1 and its analogs
First, several cell lines were used to evaluate the anti-cancer
activity of B1 (data not shown). The anti-cancer effects against
MCF-7 and K562 cell lines were comparable, while B1 was less
cytotoxic to the normal GES-1 cell line. Thus, B1 exerts selective
cytotoxicity to tumor cells while sparing healthy cells. Moreover,
B1-Ala exhibited increased anti-cancer activity over B1 (Table 1).
Then, the anti-cancer activities and selectivity of other B1 analogs were screened against MCF-7, K562 and GES-1 cells. As shown
in Table 1, the peptides with nonpolar and basic amino acids at site
14 presented more favorable anti-cancer activities, while peptides
with a polar or neutral amino acid at site 14 displayed similar activity to B1; however, replacement of Ser14 by an acidic residue
weakened the anti-cancer effects. Additionally, all the analog
peptides exhibited lower toxicity to the normal GES-1 cell line.

X. Deng et al. / European Journal of Medicinal Chemistry 89 (2015) 540e548

543

Table 1
Structural characteristics and anti-cancer activities of B1 and its analogs.
Peptides

Sequence

IC50 (mM)

Mass (Da)
Chemicophysical UPLC
properties of the retention
Calculated
time (min)
amino acid at
residue 14

B1

1VKRFKKFFRKLKKSV15-NH2

Polar neutral

3.38

B1-Ala

1VKRFKKFFRKLKKAV15-NH2

Nonpolar

3.48

B1-Val

1VKRFKKFFRKLKKVV15-NH2

Nonpolar

3.69

B1-Leu

1VKRFKKFFRKLKKLV15-NH2

Nonpolar

3.84

B1-Phe

1VKRFKKFFRKLKKFV15-NH2

Nonpolar

3.74

B1-Gly

1VKRFKKFFRKLKKGV15-NH2

Polar neutral

3.25

B1-Gln

1VKRFKKFFRKLKKQV15-NH2

Polar neutral

3.31

B1-Thr

1VKRFKKFFRKLKKTV15-NH2

Polar neutral

3.44

B1-Lys

1VKRFKKFFRKLKKKV15-NH2

Basic

3.22

B1-Arg

1VKRFKKFFRKLKKRV15-NH2

Basic

3.13

B1-His

1VKRFKKFFRKLKKHV15-NH2

Basic

3.28

B1-Asp

1VKRFKKFFRKLKKDV15-NH2

Acidic

3.38

B1-Glu

1VKRFKKFFRKLKKEV15-NH2

Acidic

3.41

[MH]: 1938.26
[M2H]2: 969.64
[M3H]3: 646.76
[MH]: 1922.27
[M2H]2: 961.64
[M3H]3: 641.43
[MH]: 1950.30
[M2H]2: 975.65
[M3H]3: 650.77
[MH]: 1964.32
[M2H]2: 982.66
[M3H]3: 655.44
[MH]: 1998.30
[M2H]2: 999.65
[M3H]3: 666.77
[MH]: 1908.25
[M2H]2: 954.63
[M3H]3: 636.76
[MH]: 1979.29
[M2H]2: 990.15
[M3H]3: 660.44
[MH]: 1952.28
[M2H]2: 976.64
[M3H]3: 651.43
[MH]: 1979.33
[M2H]2: 990.17
[M3H]3: 660.45
[M2H]2: 1004.17
[M3H]3: 669.78
[M4H]4: 502.59
[MH]: 1988.29
[M2H]2: 994.65
[M3H]3: 663.44
[MH]: 1966.26
[M2H]2: 983.63
[M3H]3: 656.09
[MH]: 1980.28
[M2H]2: 990.64
[M3H]3: 660.76

Fluorouracil
ADM

3.3. Hemolytic activity

The hemolytic activity of the B1 and its analogs against rabbit
erythrocytes was determined as a major measure of peptide
toxicity toward higher eukaryotic cells. As shown in Fig. 2, all the
tested peptides exhibited little hemolytic activity even at the concentration of 160 mM.

Observed

MCF-7 K562

GES-1

MCF-7/ADM K562/ADM

[MH]: 1938.1
[M2H]2: 969.4
[M3H]3: 646.5
[MH]: 1922.4
[M2H]2: 961.8
[M3H]3: 641.0
[MH]: 1950.2
[M2H]2: 975.7
[M3H]3: 651.1
[MH]: 1964.3
[M2H]2: 982.8
[M3H]3: 655.1
[MH]: 1998.1
[M2H]2: 999.9
[M3H]3: 666.5
[MH]: 1908.4
[M2H]2: 954.6
[M3H]3: 636.7
[MH]: 1979.3
[M2H]2: 989.9
[M3H]3: 660.2
[MH]: 1952.1
[M2H]2: 976.5
[M3H]3: 651.3
[MH]: 1979.2
[M2H]2: 989.7
[M3H]3: 660.3
[M2H]2: 1004.1
[M3H]3: 670.1
[M4H]4: 502.5
[MH]: 1988.2
[M2H]2: 994.8
[M3H]3: 663.2
[MH]: 1966.4
[M2H]2: 984.2
[M3H]3: 655.8
[MH]: 1980.1
[M2H]2: 990.9
[M3H]3: 661.0

21.9

26.9

102.3

24.1

31.6

8.9

8.6

87.1

4.9

5.0

64.6

3.6

1.1

77.6

4.4

3.3

1.3

3.7

52.5

15.8

14.8

107.2

14.5

16.2

102.3

19.1

17.0

109.6

8.3

5.5

123.0

8.6

5.5

5.5

8.7

93.3

5.0

10.5

104.7

19.9

44.7

120.2

38.9

36.6

104.5

26.9
0.36

31.6
0.45

30.9
0.53 52.5

67.6

signicant variations of membrane morphology were observed

when compared to the control group. Fig. 3 shows that untreated
cells had a normal smooth surface and the cells were intact, yet the
tumor cells treated with the peptides had disrupted cell
membranes.
3.6. Localization of peptides by FITC labeling

AO/EB double staining was employed to detect the effects on

MCF-7 cell membranes of B1, B1-Leu and B1-Lys. AO and EB are
both uorescent dyes: AO penetrates the cell membrane, incorporates into DNA and makes the cells appear green; while EB
only penetrates cells with disrupted cell membranes before
inserting into DNA and making the cells appear red. As shown in
Fig. 3, the tumor cells turned red after treatment with 20 mM B1,
5 mM B1-Leu or 8 mM B1-Lys for 30 min compared with the control.

To further investigate the possible mechanism of B1 and its

analogs, FITC-labeled peptides were used to visualize uptake. The
image acquisition for MCF-7 cells was performed after treatment
with FITC-labeled peptides for 5 min and 30 min. As shown in Fig. 3,
the uorescent intensity in the cells increased with a prolonged
incubation period, indicating that FITC-labeled peptides were being
transferred into the cytoplasm. Thus, FITC-labeled peptides might
disrupt the cell membrane and then diffuse into the cytoplasm. This
result is in accordance with the data from the AO/EB staining
experiment.

3.5. SEM visualization of the cell membrane

3.7. Mitochondrial membrane potential (DJ) assessment by JC-1

Membrane disruption of MCF-7 cells was visualized by SEM.

After incubation with B1 (20 mM), B1-Leu (5 mM) or B1-Lys (8 mM),

JC-1 is a mitochondrial membrane-potential-sensitive dye. It

exists as a monomer at low concentration, while it forms J-

3.4. Detection of cell membrane variation by AO/EB staining

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X. Deng et al. / European Journal of Medicinal Chemistry 89 (2015) 540e548

observed as red uorescence. When the mitochondrial membrane

is depolarized, JC-1 is released from the mitochondria and red
uorescence is reduced. As shown in Fig. 4, red uorescence
decreased after treatment with B1, B1-Leu or B1-Lys. These results
imply that B1 and its analogs act on mitochondrial membranes and
cause depolarization.
3.8. Assessment of cytochrome C contents

Fig. 2. The curves of the hemolytic activity of the peptides for rabbit erythrocytes.

aggregates at high concentration. Both states present green uorescence, but J-aggregates also emit red uorescence. As the normal
mitochondria have high DJ, JC-1 exhibits potential-dependent
accumulation in mitochondria and forms J-aggregates that can be

In healthy cells, cytochrome C cannot penetrate the mitochondrial membrane and enter into the cytoplasm. However,
when the mitochondrial membrane is disrupted cytochrome C
release triggers activation of caspase proteases leading to
apoptosis. To further study the actions of B1 and its analogs on
mitochondria, as well as their apoptosis-inducing effects, we
assessed the cytochrome C contents in each part of the cells. We
found that B1 and its analogs promote cytochrome C release from
the mitochondria into the cytoplasm (Table 2). In addition, we
found B1 triggers cytochrome C release in a dose-dependent
manner (Fig. 4).

Fig. 3. (A)e(C). AO/EB staining and SEM images of MCF-7 cells after treatment with B1 (20 mM), B1-Leu (5 mM) and B1-Lys (8 mM), respectively. The rst panels are the AO green
uorescence images of untreated cells and cells treated with B1, B1-Leu and B1-Lys, respectively. The second panels are the corresponding EB uorescence images of the cells. The
red indicates that EB bound to DNA after entering through the disrupted cell membrane. The third panels are the SEM visualization of untreated cells and cells treated with B1, B1Leu and B1-Lys, respectively. (D) and (E) are uorescence images of MCF-7 cells treated with the FITC-labeled peptides for 5 min or 30 min. (For interpretation of the references to
color in this gure legend, the reader is referred to the web version of this article.)

X. Deng et al. / European Journal of Medicinal Chemistry 89 (2015) 540e548

545

Fig. 4. (A, D, G and J). Green uorescence of JC-1 in control, B1, B1-Leu and B1-Lys groups, respectively. (B, E, H and K). Red uorescence of JC-1 in control, B1, B1-Leu and B1-Lys
groups, respectively. (C, F, I and L). Merged images of green and red uorescence, with orange indicating the normal DJ of the mitochondrial membrane. (M). Cytochrome C
releasing effect of B1 at various concentrations. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

3.9. The anti-migratory effects of B1 and its analogs

The anti-migratory effect of B1 and its analogs against MCF-7
cells was assessed with wound-healing and Transwell migration
assays. The migratory ability of MCF-7 cells was inhibited signicantly when treated with B1 (5 mM), B1-Leu (0.5 mM) and B1-Lys
(1 mM) (Fig. 5).
3.10. Inhibitory effects of B1 and its analogs on ADM-sensitive and
-resistant cells
We evaluated the antiproliferative effects of B1 and its analogs
on ADM-sensitive and -resistant cells by the MTT assay. As shown
in Table 1, B1 and its analogs exhibited almost equal potency to both
ADM-sensitive and -resistant cells. These data suggest that B1 and
its analogs exert effective antitumor activities against drugresistant tumor cells.

3.11. B1 and its analogs reverse ADM resistance of MCF-7/ADM and

K562/ADM cells
The cytotoxicity of ADM against ADM-resistant cells was tested
in the presence or absence of the peptides, while Verapamil was
used as the positive control. As shown in Fig. 6, ADM alone had little
inhibitory effect on the ADM-resistant cells; however, combination
with the peptides or Verapamil increased the inhibitory effects of
ADM. Thus, we suggest that B1, B1-Leu and B1-Lys possess drugresistance reversal activities.
3.12. Accumulation of ADM
ADM is a uorescent substance that can be used to monitor drug
accumulation in cells [29]. The red uorescence intensity of ADM
increased after treatment with the peptides or Verapamil (Fig. 6).
Furthermore, the level of ADM in the cells was measured by

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X. Deng et al. / European Journal of Medicinal Chemistry 89 (2015) 540e548

Table 2
Content of cytochrome C in each part of the MCF-7 cells (ng).
Control
Total content
Extracellular
Cytoplasm
Mitochondria

364.2
15.7
44.1
347.8

B1
20.5
0.77
2.7
14.5

353.4
14.7
60.3
301.4

B1-Leu

20.4
1.33
5.8*
11.8*

354.8
14.9
59.9
303.8

B1-Lys
20.8
0.46
5.3**
13.8*

359.1
15.0
59.6
303.2

5-Fu
27.3
0.67
4.3**
11.0*

357.0
15.7
49.6
319.2

20.2
0.57
1.6*
8.7*

*P < 0.05, **P < 0.01 (compared to control).

spectrouorometry. As presented in Fig. 6, ADM accumulation in

ADM-resistant cells increased signicantly after incubation with
the peptides, suggesting that B1, B1-Leu and B1-Lys facilitate ADM
entry into drug-resistant cells.

4. Discussion
AMPs are ancient innate defense compounds widely distributed
across the biological world [30]. In addition to antimicrobial activity, several AMPs also possess anti-cancer activity that might be
harnessed for cancer treatment [13,31]. As a promising potential
chemotherapeutic, AMPs exhibit signicant advantages over
traditional anti-cancer agents, such as higher specicity for cancer
cells that circumvents toxicity issues as well as the ability to bypass
drug-resistance mechanisms [12,32].
B1 is an amidated derivative of BF-15, a cathelicidin-like AMP
[21]. Here, we demonstrate the anti-cancer activity of B1 for the
rst time. The Ser14 residue is located at the interface of the hydrophobic and hydrophilic faces of B1 according to a helix-wheel
plot. As amphipathy and cationicity play pivotal roles in the activities of AMPs [33], we wondered whether the replacement of Ser14
by different amino acids would alter the activity of B1. Peptides
where Ser14 was substituted by a nonpolar or basic amino acid
showed increased anti-cancer activity, while peptides where the
Ser14 was replaced by a polar, neutral or acidic residue displayed
similar or weakened anti-cancer effects, respectively. All of the
peptides showed lower cytotoxicity to the normal GES-1 cell line
compared to the cancer cell lines examined. Moreover, these peptides exhibited little hemolysis activity. B1, B1-Leu and B1-Lys
peptides were selected for further investigation.
First, the anti-cancer mechanism of B1 and its analogs were
studied. The AO/EB staining assay suggested that B1 and its analogs
exert membrane-disrupting effects on cancer cells and SEM visualization of cell morphology corroborated this suggestion. The
images showed that the MCF-7 cell membranes were impaired
after treatment with B1 and its analogs. In addition, uorescent
microscopy observation of FITC-labeled peptides revealed that B1
and its analogs penetrate the impaired cell membrane and enter
into the cytoplasm. In summary, B1 and its analogs exert effects on
the cell membrane and alter the permeability of cancer cells, which
facilitates their entry into the cells.
Mitochondria originate from the bacterial phylum, a-Proteobacteria [34]. Similar to bacteria, mitochondria carry a net negative
charge on their surface, which is a consequence of possessing a
large quantity of the anionic lipid cardiolipin [35]. After penetrating
into the cytoplasm, AMPs gain access to mitochondria and disrupt
the mitochondrial membrane resulting in the release of mitochondrial proteins, such as cytochrome C, and apoptosis [20,36,37].
A JC-1 assay showed that treatment with B1, B1-Leu and B1-Lys
altered the mitochondrial membrane potential of MCF-7 cells,
and cytochrome C contents in the mitochondria reduced after
peptide treatment while it increased in the cytoplasm. This suggests that B1 and its analogs exert anti-cancer activity by acting on
mitochondria and causing cytochrome C release. In addition, we

found that B1 promoted cytochrome C release in dose-dependent

manner.
Based on the information, we conclude that the anti-cancer
mechanism of B1 and its analogs involves three steps: (1) cell
membrane disruption resulting in a change of membrane permeability; (2) penetration into the cytoplasm by virtue of the disrupted membrane; (3) disruption of mitochondrial membranes and
release of cytochrome C.
A wound healing assay and Transwell migration assay were
applied to appraise the anti-migratory effects of B1 and its analogs
at maximal nontoxic concentrations. Each peptide exhibited
inhibitory effects on the migration of MCF-7 cells.
As conventional chemotherapeutic agents enter into the cancer
cells before exerting their action, cells can develop resistance by
pumping the drugs out via multidrug-resistant proteins [38]. The
unique membrane-disrupting mechanism of AMPs against cancer
cells meant that we were interested in investigating the antiproliferative and drug-resistance reversing effects on ADMresistant cells. In this present study, we found that ADM-sensitive
and -resistant cells were equally susceptible to B1 and its analogs.
This is mainly due to the net positive charge and amphipathy of the
peptides which contribute to the membrane disruption of cancer
cells, including drug-resistant cells. B1 and its analogs also exerted
drug resistance-reversing activities against ADM-resistant cancer
cell lines. An ADM accumulation assay conrmed that the contents
of ADM in MCF-7/ADM or K562/ADM cells were increased

Fig. 5. Anti-migratory assay of B1 and its analogs. In the wound-healing assay, the
upper panels are images of untreated cells and cells treated with B1 (5 mM), B1-Leu
(0.5 mM), B1-Lys (1 mM) and 5-Fu (10 mM) at 0 h, respectively; lower panels are corresponding images at 24 h.

X. Deng et al. / European Journal of Medicinal Chemistry 89 (2015) 540e548

547

Fig. 6. ADM resistance-reversing effects and ADM accumulation assay of B1 and its analogs. Fluorescence images: (A) and (F) are images of MCF-7/ADM or K562/ADM cells treated
with 20 mM ADM, respectively. (B) and (G) [(C) and (H), (D) and (I), or (E) and (J)] are the images of MCF-7/ADM or K562/ADM treated with 10 mM Verapamil (5 mM B1, 0.5 mM B1-Leu
or 1 mM B1-Lys) and 20 mM ADM, respectively.

signicantly when incubated with the peptides. We deduce that B1

and its analogs affect the permeability of the cell membrane and
promote greater diffusion of agents into the cells. Data from EB
staining and FITC-peptide assays also indirectly conrm this
assertion. Thus, B1 and its analogs overcome drug-resistance by
disrupting the cell membrane and permitting more drugs to
permeate into drug-resistant cells.
At present, it has been reported a great number of AMPs exert
selective cytotoxicity to cancer cells, including the drug-resistant
ones [12,13]. Compare to these peptides, B1 and its analogs present the similar anti-cancer activity against the neoplastic cells.
Notably, B1 and its analogs also exhibit drug-resistance reversing
and anti-migratory effects, which have not been reported on other
AMPs. Therefore, that might provide a new idea for designing AMPs
with drug-resistance reversing as well as anti-migratory activity.

nonpolar and basic amino acid had increased anti-cancer activity

over B1 and maintained specicity for cancer cells. These peptides
exerted their anti-cancer activity by disrupting the cell membrane
and entering into the cytoplasm, before acting on the mitochondria
and stimulating the release of cytochrome C. Additionally, these
peptides also prevented the migration of cancer cells. B1 and its
analogs remained potent against drug-resistant cell lines and they
also exerted drug resistance-reversing effects against MCF-7/ADM
and K562/ADM cells. Besides, B1 and its analogs showed lowhemolytic activity. Therefore, we suggest that B1 analogs might
be promising candidates for cancer therapy.

5. Conclusion

Acknowledgments

In summary, we report the anti-cancer activity of peptide B1 for

the rst time. We then designed and synthesized a series of B1
analogs. Peptides where the Ser14 residue was substituted by a

The work was supported by the National Natural Science

Foundation of China (No. 81172932 and 81273376), the Natural
Science Foundation of Jiangsu Province (No. BK2012356).

Conict of interest
The authors have no conicts of interest to declare.

548

X. Deng et al. / European Journal of Medicinal Chemistry 89 (2015) 540e548

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