Antineoplastic activity of linear leucine homodipeptides and their potential mechanisms of action

Article abstract of DOI:10.1097/CAD.0000000000000615

Galaxamide is a rare cyclic homopentapeptide composed of three leucines and two N-methyl leucines isolated from marine algae Galaxaura filamentosa. The strong antitumor activity of this compound makes it a promising candidate for tumor therapy. The synthesis of galaxamide, however, is a complex process, and it has poor water solubility. On the basis of its special chemical composition, we designed a series of linear leucine homopeptides. Among seven dipeptide derivatives, five compounds with terminal protection groups and methyl substitution of the hydrogen in the amido group showed remarkable inhibitory effects against various cancer cells. N-Tertbutyl-d-leucine-N-methyl-d-leucinebenzyl (A7), the only stereomer condensed by two d-leucines, showed the highest antineoplastic activity. A7-Treated cells showed cell cycle arrest and morphological changes typical of cells undergoing apoptosis. The population of Annexin-V positive/propidium iodide-negative cells also increased, indicating the induction of early apoptosis. A7 promoted the cleavage of caspase-9 and caspase-3, as well as increased intracellular Ca 2+ levels and decreased the mitochondrial membrane potential. Collectively, certain linear leucine dipeptides derived from cyclic pentapeptide are able to inhibit tumor cell proliferation through cell cycle arrest and apoptosis induction. The N-methyl group in the side chain and the d/l conformation of the amino-Acid residue are critical for their activity.

Full text of DOI:10.1097/CAD.0000000000000615

Preclinical report
1
Antineoplastic activity of linear leucine homodipeptides and
their potential mechanisms of action
Yun Leia, Xiao-Xia Yangb,d, Wei Guob, Fu-yong Zhangb, Xiao-Jian Liaob,
Hui-Fu Yangb, Shi-Hai Xub and Sheng Xionga,c
Galaxamide is a rare cyclic homopentapeptide composed of
three leucines and two N-methyl leucines isolated from
marine algae Galaxaura filamentosa. The strong antitumor
activity of this compound makes it a promising candidate
for tumor therapy. The synthesis of galaxamide, however, is
a complex process, and it has poor water solubility. On the
basis of its special chemical composition, we designed a
series of linear leucine homopeptides. Among seven
dipeptide derivatives, five compounds with terminal
protection groups and methyl substitution of the hydrogen
in the amido group showed remarkable inhibitory effects
against various cancer cells. N-tertbutyl-D-leucine-N-methyl-
D-leucinebenzyl (A7), the only stereomer condensed by two
D-leucines, showed the highest antineoplastic activity.
A7-treated cells showed cell cycle arrest and morphological
changes typical of cells undergoing apoptosis. The
cyclic pentapeptide are able to inhibit tumor cell
proliferation through cell cycle arrest and apoptosis
induction. The N-methyl group in the side chain and the D/L
conformation of the amino-acid residue are critical for their
activity. Anti-Cancer Drugs 00:000–000 Copyright © 2018
Wolters Kluwer Health, Inc. All rights reserved.
Anti-Cancer Drugs 2018, 00:000–000
Keywords: antineoplastic activity, apoptosis, cyclic peptide,
homo-oligopeptide, leucine dipeptide
aDepartment of Cell Biology, Institute of Biomedicine, bDepartment of Chemistry,
Jinan University, cNational Engineering Research Center of Genetic Medicine,
Guangzhou and dZhaoqing Aquatic Technologies Popolarizing Center, Zhaoqing,
People’s Republic of China
Correspondence to Sheng Xiong, PhD, Department of Cell Biology, 730 Building
of Biology, Jinan University, 601 W Huangpu Avenue, Guangzhou 510632,
Guangdong, People’s Republic of China
Tel/fax: + 86 208 522 2759; e-mail: xsh_jnu@hotmail.com
population of Annexin-V positive/propidium iodide-negative
cells also increased, indicating the induction of early
apoptosis. A7 promoted the cleavage of caspase-9 and
caspase-3, as well as increased intracellular Ca2 + levels
and decreased the mitochondrial membrane potential.
Collectively, certain linear leucine dipeptides derived from
or
Correspondence to Shi-Hai Xu, PhD, Department of Chemistry, Jinan University,
Guangzhou 510632, Guangdong, People’s Republic of China
Tel/fax: + 86 208 522 8856; e-mail: txush@jnu.edu.cn
Received 3 October 2017 Revised form accepted 12 February 2018
Introduction
angiogenesis inhibition, stimulation of immune response,
or blocking tumor-related processes such as cell adhesion
and metastasis through various pathways [5]. Peptides
from natural sources can either serve as therapeutic agents
directly or as ‘lead’ compounds for further modification to
improve their therapeutic potential [6]. Efforts have been
focused on the artificial synthesis of such peptide-based
compounds and development of their derivatives with
improved biological activity. Compared with linear
counterparts, cyclic peptides have advantages because of
their exceptional stability and enhanced activities [7].
However, laboratory synthesis of cyclic peptides is still
challenged by several thermodynamic and kinetic issues.
Therefore, most peptides are naturally synthesized as
linear chains.
Now, therapeutic antibodies have emerged to be the star
category of anti-cancer drugs [1]. However, the complex
structure and large molecular size make the manufacture
and utilization of antibodies difficult. For example, because
of their large size, antibodies have a decreased ability to
penetrate tissues and cannot pass through the blood–brain
barrier. These factors limit their usage in diseases of the
central nervous system [2]. Small peptides from natural
sources have certain competitive advantages over small-
molecule drugs and monoclonal antibodies because of their
small size, low toxicity and immunogenicity, strong target
specificity, and the relative ease with which they can be
modified and designed [3,4]. Thus, they have attracted
considerable attention as candidates for cancer therapy.
Small peptides with antineoplastic activity can be found
in sources such as terrestrial animals, marine organisms,
microorganisms, milk, and eggs. They exert their
tumor-inhibitory effects through apoptosis induction,
Galaxamide is a monocyclic pentapeptide that we first
isolated from marine algae Galaxaura filamentosa. In a
previous study, we designed a retrosynthesis strategy and
successfully synthesized this cyclic peptide. An in-vitro
cytotoxic assay showed that it was biologically active
against the human renal cell carcinoma GRC-1 cells
and human hepatocellular carcinoma HepG2 cells, with
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DOI: 10.1097/CAD.0000000000000615
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2
Anti-Cancer Drugs 2018, Vol 00 No 00
corresponding IC50 values of 4.26 and 4.63 μg/ml [8]. The
antitumor potential of this cyclic pentapeptide makes it a
promising candidate for tumor therapy; however, the
complex process required for synthesis and the relatively
poor water solubility of this compound are limitations. As
a rare cyclopeptide, galaxamide is highly symmetric in
structure. It is composed of three leucines and two
N-methyl leucines, which contribute toward its insolu-
bility. On the basis of its chemical composition, we
explored the possibility that some linear oligopeptides
derived from galaxamide would also be biologically active
against tumors while having a smaller molecular size and
better water solubility. Following this idea, a series of
leucine homo-oligopeptides were synthesized and eval-
uated. Interestingly, certain leucine homo-oligopeptides
including dipeptides, tripeptides, and pentapeptides
showed antineoplastic activity. The dipeptides were
especially active, and some inhibited tumor cell pro-
liferation to a degree comparable to that of galaxamide in
certain tumor cell lines. In this paper, we summarized the
synthesis, structure, antineoplastic activity, and potential
mechanisms of action of the novel leucine dipeptides.
and a saturation aqueous solution of NH4Cl (10 ml) and
saturation NaHCO3 was added in turn. The THF was
evaporated in decompression. The aqueous solution
were washed with petroleum ether (2 × 25 ml), 1 mol HCl
was added to adjust the pH as 2–3, and extracted with
EtOAc (4 × 50 ml). The combined organic layers were
washed with sodium thiosulfate (5%, 75 ml) and satura-
tion NaCl (75 ml), dried (Na2SO4), filtered, and con-
centrated. Finally, a 1.99 g product was obtained. Yellow
oil, yield 87%, 1H NMR (400 MHz, CDCl3) δ (ppm): 4.82
3
2
(m, 0.5H), 4.62 (m, 0.5H), 2.80 (s, H), 1.71 (m, H), 1.58
1
9
6
(m, H), 1.46 (s, H), 0.95 (d, J = 6.4 Hz H); MS (ESI)
m/z: 246.2 [M + H]+, 263.2 [M + NH4]+, 268.3 [M + Na]+.
Leu-OBn (E6)
The reaction was started with L-Leu (30 mmol, 3.9 g),
benzyl methanol (45 mmol, 4.8 ml), P-toluenesulfonic
acid (36 mmol, 60 g), and toluene (90 ml) and heated to
reflux for 4 h, cooled and diluted by aether (80 ml),
washed with saturation sodium carbonate (2 × 80 ml), and
the aqueous layers were extracted with aether (2 × 80 ml).
The combined organic layers were dried (Na2SO4),
filtered, the solvent was evaporated, and repeated crystal
by EtOAc. Finally, 9.76 g L-Leu-OBzl·TosOH was
Materials and methods
1
Peptide synthesis, purification, and characterization
All reagents were obtained from commercial suppliers and
used without further purification, except for the evaporation of
methanol, tetrahydrofuran (THF), and dichloromethane. The
reactions were carried out under an inert atmosphere of N2,
and the progress of the reaction was monitored by thin-layer
chromatography on precoated glass plates (silica gel 60, F254,
0.25 mm, Ocean Chemical Company, Qingdao, China). High-
performance liquid chromatography was used to determine
the purity of certain compounds. The melting points were
measured by a thermometer without calibration. Optical
obtained, yield 82%, melting point (m.p.): 54–58°C; H
NMR (500 MHz, CDCl3) δ (ppm): 7.77 (d, J = 8.1 Hz
2H), 7.33 (m, 5H), 7.18 (d, J = 8.0 Hz 2H), 5.22 (dd, J = 12,
2
1
3
24.3 Hz H), 3.84 (m, H), 2.73 (t, J = 5.2, H), 2.36 (s,
2H), 1.87 (m, H), 1.68 (m, H), 0.87 (m, H).
2
1
6
Synthesis of Leu–Leu dipeptide benzyl ester derivatives
Boc-L-Leu-N-Me-L-Leu-OBn (A1)
A reaction mixture containing Boc-L-Leu-OH (4.8 mmol,
1.1 g) in THF (8 ml), DEPBT (6.0 mmol, 1.8 g), and DIEA
(6.0 mmol, 1.05 ml) was stirred at 0°C for 5 min. Then,
H-N-Me-L-Leu-OBn·TosOH (4 mmol, 1.6 g) was added and
raised to room temperature naturally and stirred vigorously
for 8 h. The solvent was evaporated in decompression. The
residue was purified by column chromatography (silica gel,
hexane/acetone =20 : 1). Finally, a 1.76 g product was
obtained. Achromaticity crystalloid, yield 89%, m.p.: 66–68°
C; [α]D24–67 (c=0.19 CH3OH); 1H NMR (500 MHz,
DMSO) δ (ppm): 7.35 (m, 5H), 6.97 (d, J=8.0, 1H), 5.23 (dd,
J=11.3, 3.0 Hz, 1H), 5.09 (m, 2H), 4.33 (m, 1H), 2.89 (s, 3H),
rotations were measured using
a Schmidt +Haensch
Polartromic instrument (Schmidt +Haensch, Berlin,
D
Germany). Proton and carbon NMR spectra were recorded on
an Inova 500NB: Varian, Palo Alto, California, USA
(500 MHz). Liquid chromatography–mass spectrometry
(LCMS) of the synthesized compounds was examined on an
LCMS-4000 Q TRAP Shimadzu spectrometer (Applied
Biosystems, Waltham, Massachusetts, USA) using the elec-
trospray ionization (ESI) mode of ionization. The three-
dimensional configuration space of A7 was determined by
X-ray crystallography (Smart 1000 CCD; Bruker: Billerica,
Massachusetts, USA).
1
2
1
9
1.76 (m, H), 1.58 (m, H), 1.45 (m, H), 1.34 (s, H), 1.20
(m, 2H), 0.93 (m, 12H); 13C NMR (CDCl3, 125 Hz) δ (ppm):
173.9, 171.5, 155.7, 135.4, 128.5 (2C), 128.3, 128.0 (2C), 79.4,
66.9, 54.4, 48.9, 41.9, 36.8, 30.7, 28.2 (3C), 24.7, 24.5, 23.3,
23.2, 21.7, 21.3; MS (ESI) m/z: 449.3 [M +H]+, 466.5
[M +NH4]+, 471.6 [M +Na]+.
Synthesis of two material segment
N-Me-Boc-Leu (E3)
Hydriodic methane (15 mmol, 3.15 ml) was added to a
solution of Boc-L-Leu-OH (10 mmol, 2.31 g) in THF
(30 ml) slowly under an ice bath. The mixture was stirred
vigorously for 30 min and 60% hydrid sodium (50 mmol,
1.2 g) was added smoothly. The mixture was stirred for
1h more in ice bath, and then be reacted overnight in
room temperature. The reaction was cooled in an ice bath
BOC-N-Me-L-Leu–L-Leu-OBn (A2)
A reaction mixture containing Boc-N-Me-L-Leu-OH
(6 mmol, 1.47 g) in THF (8 ml), DEPBT (9.0 mmol,
2.69 g), and DIEA (9.0 mmol, 1.6 ml) was stirred at 0°C
for 5 min. Then, L-Leu-OBn·TosOH (6.6 mmol, 2.59 g)
was added and raised to room temperature naturally and
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Antineoplastic activity of Leu dipeptide Lei et al.
3
stirred vigorously for 12 h. The solvent was evaporated in
decompression. The residue was purified by column
chromatography (silica gel, hexane/acetone = 20 : 1).
Finally, a 2.42 g product was obtained. Achromaticity
crystalloid, yield 90%, [α]D24–64 (c = 0.26, CH3OH); m.
Then, L-Leu-OBn·TosOH (6 mmol, 2.4 g) was added and
raised to room temperature naturally and stirred vigorously
for 6 h. The solvent was evaporated in decompression.
The residue was purified by column chromatography
(silica gel, hexane/acetone= 20:1). Finally, a 2.47 g product
was obtained. Achromaticity crystalloid, yield 87%, [α]
1
p.: 68–69°C; H NMR (500 MHz, CDCl3) δ (ppm): 7.36
5
1
2
2
1
(m, H), 6.51 (s, H), 5.16 (m, H), 4.65 (s, H), 2.75
D24 + 9.6 (c = 0.32, CH3OH); m.p.: 51–52°C; H NMR
3
4
2
9
(s, H), 1.63 (m, H), 1.54 (m, H), 1.48 (s, H), 0.93 (m,
12H); MS (ESI) m/z: 449.3 [M + H]+, 466.5 [M + NH4]+,
471.6 [M + Na]+.
(CDCl3) δ (ppm): 7.31 (m, 5H), 5.25 (d, 1H), 5.14 (m, 3H),
4.70 (m, 1H), 2.97 (s, 3H), 1.71 (m, 4H), 1.44 (s, 11H), 1.33
(m, 2H), 0.93 (m, 12H); 13C NMR (CDCl3) δ (ppm): 173.9
(1C), 171.5 (1C), 155.7 (1C), 135.4 (1C), 128.5 (1C), 128.4
(1C), 128.3 (1C), 128.2 (2C), 79.4 (1C), 66.9 (1C), 54.4 (1C),
48.9 (1C), 41.9 (1C), 36.9 (1C), 30.8 (1C), 28.3 (1C), 28.2
(1C), 28.2 (1C), 24.7 (1C), 24.5 (1C), 23.3 (1C), 23.2 (1C),
21.7 (1C), 21.3 (1C). MS (ESI) m/z: 449.3 [M+ H]+, 466.5
[M+ NH4]+, 471.6 [M+ Na]+.
Boc-L-Leu-L-Leu-OBn (A3)
A reaction mixture containing Boc-L-Leu-OH (6.6 mmol,
1.5 g) in THF (10 ml), DEPBT (9.0 mmol, 2.69 g), and
DIEA (9.0 mmol, 1.6 ml) was stirred at 0°C for 5 min.
Then, L-Leu-OBn·TosOH (6 mmol, 2.4 g) was added
and raised to room temperature naturally and stirred
vigorously for 6 h. The solvent was evaporated in
decompression. The residue was purified by column
chromatography (silica gel, hexane/acetone = 20:1).
Finally, a 2.47 g product was obtained. Achromaticity
crystalloid, yield 92%, [α]D24–31 (c = 0.25, CH3OH); m.
Boc-N-Me-L-Leu-N-Me-L-Leu-Obn (A6)
A solution of Boc-L-Me-Leu-OH (3.3 mmol, 0.81 g) in
THF (5 ml) was prepared under an ice bath, and DEPBT
(4.5 mmol, 1.35 g) and DIEA (4.5 mmol, 0.8 ml) were
added in turn. After stirring for 5 min, H-N-Me-L-Leu-
OBn·TosOH (3 mmol, 1.27 g) was added to the mixture,
raised to room temperature naturally, and stirred vigorously
for 18 h. The solvent was evaporated in decompression.
The residue was purified by column chromatography (silica
gel, hexane/acetone =20:1). Finally, a 1.12 g product was
obtained. Achromaticity oil, yield 81%, [α]D24–109.6
(c = 0.7, CH3OH); 1H NMR (CDCl3) δ (ppm): 7.33 (m, 5H),
5.31 (m, 1H), 5.09 (m, 2H), 4.85 (m, 1H), 2.83 (s, 3H), 2.70 (s,
3H), 1.70 (m, 4H), 1.43 (s, 9H), 1.37 (m, 2H), 0.91 (m, 12H);
MS (ESI) m/z: 463.3 [M +H]+, 480.5 [M +NH4]+, 485.6
[M + Na]+.
1
p.: 86–87°C; H NMR (500 MHz, CDCl3) δ (ppm): 7.35
5
1
2
(m, H), 6.40 (d, J = 7.9 Hz, H), 5.16 (m, H), 4.86 (d,
1
1
1
4
J = 7.5 Hz, H), 4.67 (m, H), 4.09 (s, H), 1.68 (m, H),
1.56 (m, H), 1.44 (s, H), 0.91 (m, 12H) MS (ESI) m/z:
435.3 [M + H]+, 452.5 [M + NH4]+, 457.6 [M + Na]+.
2
9
Boc-D-Leu-N-Me-L-Leu-OBn (A4)
Boc-D-Leu-OH (11 mmol, 2.54 g) was dissolved in
dichloromethane (80 mL) in ice bath; HOBt (11 mmol,
1.47 g) and EDCl (11 mmol, 2.10 g) were added in turn,
and dripted DIEA (11 mmol, 1.9 ml). After stirring for
20 min, H-N-Me-L-Leu-OBn·TosOH (10 mmol, 4.07 g)
was added to the mixture, raised to room temperature
naturally, and stirred vigorously for 24 h. The reaction
was cooled in an ice bath and a half-saturation aqueous
solution of NH4Cl (3 × 50 ml) was added, washed with
saturation brine (50 ml), dried (Na2SO4), filtered, and
concentrated. The residue was purified by chromato-
graphy (silica gel, hexane/acetone = 20 : 1). Finally, a 3.9 g
product was obtained. Achromaticity crystalloid, yield
87%, [α]D24–7.5 (c = 0.21, CH3OH); m.p.: 49–49.9°C; 1H
Boc-D-Leu-N-Me-D-Leu-Obn (A7)
A solution of H-N-Me-D-Leu-OBzl·TosOH (4 mmol, 1.6 g)
was prepared in dichloromethane (50 ml) under an ice bath;
water (40 ml), Boc-D-Leu-OH (4 mmol, 0.924 g), and HOBt
(4 mmol, 0.54 g) were added in turn. The mixture was
stirred for 30 min at 0–5°C, EDCl (4.4 mmol, 0.84 g) was
added, and stirred for another 24 h at 0–5°C. Then, a
diluted aqueous solution of HCl (0.5 mol, 20 ml) was added.
The organic and the aqueous layers were separated and the
latter were extracted with EtOAc (3 × 3 ml). The combined
organic layers were washed with saturation NaHCO3 three
times and saturation citric acid two times, and then washed
to litmusless pH by saturation NaCl, dried (Na2SO4),
filtered, and concentrated. The residue was purified
by chromatography (silica gel, hexane/acetone =30 : 1).
Finally, a 1.54 g product was obtained. Achromaticity crys-
talloid, yield 86%, [α]D20–0.70 (c =0.2 CH3OH); m.p.:
49–49.9°C. 1H NMR (DMSO) δ (ppm): 7.35 (m, 5H), 6.94
(d, J=8.5 Hz, 1H), 5.10 (s, 2H), 5.22 (dd, J=4.5, 11 Hz, 1H),
5
NMR (CDCl3) δ (ppm): 7.32 (m, H), 5.24 (d, J = 9.0
Hz, 1H), 5.15 (m, 3H), 4.69 (m, 1H), 2.97 (s, 3H), 1.73 (m,
9
2
4H), 1.42 (s, H), 1.37 (m, H), 0.92 (m, 12H); 13C NMR
(CDCl3, 125 Hz), δ (ppm): 173.9 (1C), 171.3 (1C), 155.5
(1C), 135.6 (1C), 128.7 (1C), 128.5 (1C), 128.2 (1C), 128.0
(2C), 79.4 (1C), 66.8 (1C), 55.2 (1C), 49.14 (1C), 42.8 (1C),
37.4 (1C), 31.6 (1C), 28.3 (3C), 25.0 (1C), 24.6 (1C), 23.3
(1C), 23.2 (1C), 21.9 (1C), 21.3 (1C). MS (ESI) m/z: 449.3
[M + H]+, 466.5 [M + NH4]+, 471.6 [M + Na]+.
1
3
1
2
Boc-L-Leu-N-Me-D-Leu-Obn (A5)
4.43 (m, H), 2.97 (s, H), 1.82 (m, H), 1.59 (m, H), 1.45
2
9
1
A reaction mixture containing Boc-L-Leu-OH (6.6 mmol,
1.5 g) in THF (10 ml), DEPBT (9.0 mmol, 2.69 g), and
DIEA (9.0 mmol, 1.6 ml) was stirred at 0°C for 5 min.
(m, H), 1.35 (m, H), 1.23 (m, H), 0.86 (m, 12H); 13C
NMR (CDCl3, 125 Hz) δ (ppm): 173.96 (1C), 171.31 (1C),
155.57 (1C), 135.65 (1C), 128.71 (1C), 128.59 (1C), 128.26
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4
Anti-Cancer Drugs 2018, Vol 00 No 00
(1C), 128.08 (2C), 79.42 (1C), 66.81 (1C), 55.29 (1C), 49.14
(1C), 42.85 (1C), 37.40 (1C), 31.66 (1C), 28.35 (3C), 25.05
(1C), 24.65 (1C), 23.39 (1C), 23.26 (1C), 21.90 (1C), 21.30
(1C). MS (ESI) m/z: 449.3 [M+H]+, 466.5 [M+NH4]+,
471.6 [M +Na]+.
Cell cultures
The human hepatoma BEL-7402 cells, breast cancer MCF-
7 cells, human cervical epithelial carcinoma HeLa cells,
hepatoma HepG2 cells, and human lung carcinoma A549
cells were obtained from the Department of Pathology of
Fig. 1
The chemical synthesis of novel leucine dipeptide benzyl ester derivatives. (a) The chemical structures of galaxamide. (b) The chemical structures of
E1–E6. E1–E3 are isomers of Boc-Leu and E4–E6 are isomers of Leu-Bn. (c) A schematic representation of the strategies for the condensation
reactions. (d) The chemical structures of A1–A7: A1, N-tertbutyl-L-leucine-N-methyl-L-leucinebenzyl; A2, N-tertbutyl-L-leucine-L-leucinebenzyl;
A3, N-methyl-tertbutyl-L-leucine-L-leucinebenzyl; A4, N-tertbutyl-D-leucine-N-methyl-L-leucinebenzyl; A5, N-tertbutyl-L-leucine-N-methyl-D-
leucinebenzyl; A6, N-methyl-tertbutyl-L-leucine-N-methyl-L-leucinebenzyl; A7, N-tertbutyl-D-leucine-N-methyl-D-leucinebenzyl.
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Antineoplastic activity of Leu dipeptide Lei et al.
5
Table 1 The IC50 values of A1–A7 against human cancer cell lines
IC50 (μg/ml)
Compounds
HepG2
MCF-7
Bel-7402
HeLa
A549
5-FU
A1
A2
A3
A4
A5
A6
A7
29.43 2.31
15.56 1.03
> 100
1.10 0.11
17.32 0.98
> 100
16.89 0.76
11.56 1.20
> 100
2.13 0.31
> 100
> 100
> 100
> 100
> 100
10.7 0.69
> 100
5.47 0.28
> 100
> 100
> 100
> 100
> 100
8.09 0.34
> 100
> 100
> 100
> 100
8.36 0.47
8.46 0.26
8.09 0.33
4.57 0.19
12.04 0.81
12.73 0.94
8.84 0.31
6.33 0.22
9.16 0.35
9.74 0.78
15.98 1.01
6.17 0.39
5-FU, 5-fluorouracil.
Cell cycle analysis
Fig. 2
HepG2 cells were seeded in a six-well plate at a density
of 1 × 106 cells/ml. After exposure to drugs, the cells were
harvested by centrifugation, fixed with ice-cold 75%
ethanol, and then resuspended in binding buffer. The
cells were subsequently treated with RNase A for 30 min
and stained with propidium iodide (100 μg/ml) at 37°C
for 30 min in the dark. The stained cells were then ana-
lyzed by a flow cytometry system (Coulter Epics Elite,
Beckman Coulter, California, USA). The percentage of
cells in sub-G1 phase, G1 phase, S phase, and G2 phase
was analyzed using standard ModiFit (Verity Software
House, Topsham, Maine, USA) and CellQuest software
programs (BD Biosciences, San Jose, California, USA).
Apoptosis assay
The cells were treated with various concentrations of A7
(0, 6.25, 12.5, and 25.0 μg/ml) for 24 h or with a fixed
concentration of A7 (12.5 μg/ml) for different durations of
time (0, 12, 24, or 48 h). After collecting and washing with
PBS, the cells were resuspended in 500 μl of Annexin-V
binding buffer. After adding Annexin-V-FITC/PI, cells
were incubated in the dark for 15 min, acquired on a flow
cytometry system, and analyzed using CellQuest soft-
ware (WinMDI 2.9; BD Biosciences, San Jose, California,
USA). The fraction of the cell population in different
quadrants was analyzed using quadrant statistics.
A7 treatment reduces tumor cell proliferation. HepG2 cells were incubated
with different concentrations of A7 for 24, 48, and 72 h, and cell
proliferation was determined using the MTT assay. Cells treated with Eto
and 5-fluorouracil (5-FU) for 72 h were used as a control. The experiments
were repeated three times. Data are presented as mean SD, N=4.
Jinan University (Guangzhou, China). The cells were cul-
tured in RPMI-1640 medium (GIBCO, Grand Island, New
York, USA) containing 10% neonatal bovine serum, 100 U/
ml penicillin, and 100 μg/ml streptomycin. The cells were
cultured at 37°C in a 5% CO2, 95% air incubator, and fed
every 2–3 days.
Evaluation of morphological changes of HepG2 cell by
atomic force microscopy
HepG2 cells were treated with A7 (0, 12.5, and 25.0 μg/
ml) for 24 h and subsequently fixed with 2.5% glutar-
aldehyde for 30 min at 4°C. The cells were then washed
twice with cold PBS, air-dried, and observed under an
atomic force microscope (Thermo Microscopes, Thermo
Scientific, Waltham, Massachusetts, USA).
Cell proliferation assay
Cells in the logarithmic growth phase were harvested and
seeded at a density of 4 × 104 cells/ml in 96-well plates
(Costar, Cambridge, Massachusetts, USA) overnight.
The stock solutions of synthesized peptides were dis-
solved in DMSO at concentrations of 25 mg/ml. When
used, the stock solutions were doubly diluted from 100.0
to 3.125 μg/ml by RPMI-1640 medium containing 3%
neonatal bovine serum. After treating the cells with var-
ious concentrations of the compounds for 72 h, the
cytotoxicity of the compounds was determined by the
MTT (Sigma, St Louis, Missouri, USA).
AO staining of the nuclei of A7-treated HepG2 cells
HepG2 cells were treated with A7 (0, 12.5, and 25.0 μg/
ml) for 24 h. Then, the cells were collected, washed with
PBS, and resuspended in 1 ml acridine orange (100 μg/ml
acridine orange in PBS). After incubation for 15 min, the
cells were examined under a fluorescence microscope
(400-fold amplification) (Nikon, Tokyo, Japan).
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6
Anti-Cancer Drugs 2018, Vol 00 No 00
Fig. 3
(a)
(b)
(d)
(c)
A7 induces cell cycle arrest and apoptosis. (a) The cell cycle distribution. Number CICCPs in bold and italics correspond to significant variations
compared with the control. (b) Annexin-V/PI staining for apoptotic cells. Numbers in bold correspond to significant variations compared with the
control. (c) Morphological observations of apoptotic cells. Apoptotic nuclei (blue arrows), microvilli (black arrows), rough endoplasmic reticulum and
mitochondria (red arrow), nuclei (N), nucleolus (NL), and apoptotic body (hollow triangles) are indicated. (d) Atomic force microscopy (AFM)
observation. Subpanel 1 shows the topological morphology of a single representative cell, bar: 1 mm. Subpanel 2 shows the line profile of subpanel 1
along the A and B labeled line. Subpanel 3 shows the ultrastructure of the cell membrane surface zoomed from the left images, box bar: 100 nm.
Subpanel 4 shows the histogram of the particles of subpanel 3. CICCP, carbonyl cyanide-m-chlorophenylhydrazone; PI, propidium iodide.
Transmission electron microscopy of HepG2 cells
tetracetate and lead citrate, the cells were examined by
JEM-100CXII transmission electron microscopy (Jeol Ltd,
Tokyo, Japan).
HepG2 cells were treated with 0, 12.5, or 25.0 μg/ml of A7
for 24 h. The cells were harvested, washed twice with cold
PBS, and fixed with 2.5% glutaraldehyde in 0.1 mol/l PBS
(pH 7.2) at 4°C. Then, the cells were postfixed with
osmium tetroxide, dehydrated in an ethanol series, and
embedded in epoxy resin. After staining with uranium
Western blotting assay
Cells treated with 6.25–25.0 µg/ml of A7 for 24 h
were lysed in lysis buffer (Beyotime Institute of
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Antineoplastic activity of Leu dipeptide Lei et al.
7
Fig. 4
Total caspase activity assays
HepG2 cells (1 × 106 cells/ml) were harvested and
washed twice with PBS. The cell lysate was incubated
with the colorimetric substrate of caspase-8 or caspase-3
in a 96-well plate at 37°C for 4 h in a CO2 incubator.
Absorbance at 405 nm was determined by reading the
plate with a microplate reader. Activities of caspase-8 and
caspase-3 were expressed as the optical density value.
Intracellular Ca2 + concentration and mitochondrial
membrane potential
Following appropriate treatment, the cells were collected
by centrifugation, washed, and incubated at 37°C with
Fluo-3 AM for 40 min or rhodamine-123 for 20 min. The
cells were then washed three times with cold PBS. The
intracellular Ca2 + concentration and the mitochondrial
membrane potential (ΔΨm) were measured by flow cyto-
metry. Carbonyl cyanide-m-chlorophenylhydrazone (Acros
Organics, Geel, Belgium) was used as a positive control.
The results were analyzed using Orig. 6.0 software
(OriginLab Corporation, Northampton, Massachusetts,
USA).
Statistical analysis
Data are presented as mean SD. Comparisons between
groups were performed using the analysis of variance test.
Values of P less than 0.05 were considered to be statis-
tically significant.
Results
Design and synthesis of novel leucine dipeptide benzyl
ester derivatives
To screen for leucine dipeptides with antitumor activity,
we first designed six leucine derivatives (E1–E6), which
were different from each other in the benzyl (Bn) or tert-
butyloxycarbonyl (Boc) protective groups, the methyl
group in amido, and D/L stereochemistry (Fig. 1b).
Previously, we synthesized A1, N-tertbutyl-L-leucine-
N-methyl-L-leucinebenzyl, by coupling E5 and E1 [9]. In
the present study, six novel leucine dipeptide benzyl
ester derivatives (A2–A7) were designed and synthesized
by alternatively using E1–E3 and E4–E6 for condensa-
tion reactions (Fig. 1c and d). The three-dimensional
conformation of A7 was studied by X-ray crystallographic
analysis, which confirmed that this compound was not
racemized during chemical synthesis (Supplementary
Fig. S1, Supplemental digital content 1, http://links.lww.
com/ACD/A251 and Fig. S2, http://links.lww.com/ACD/
A252).
Activation of the caspase cascade. (a) Western blots were performed to
detect cleaved caspase-9 and caspase-3. (b) Colorimetric assay to
detect the total activity of caspase-8 and caspase-3. Data are presented
as mean SD (N = 3). *P < 0.05 versus the control group.
Biotechnology, Jiangsu, China) and centrifuged to collect
cell extracts for electrophoresis. Proteins (50 µg) from
samples were resolved on 10% SDS-polyacrylamide gels
and transferred to epolyvinylidene difluoride mem-
branes. After blocking with 5% nonfat milk, the mem-
branes were probed with mouse anti-human-caspase
monoclonal antibodies (Cell Signaling Technology,
Boston, Massachusetts, USA) and a rabbit anti-GAPDH
polyclonal antibody (Santa Cruz Biotechnology, Santa
Cruz, California, USA). They were subsequently probed
with peroxidase-conjugated secondary antibodies (KPL,
Gaithersburg, Maryland, USA). Detection was carried out
using the SuperSignal West Pico kit (Pierce Biotechnology,
Rockford, Illinois, USA).
Antitumor activity of A1–A7 in human cancer cells
The anticancer activity of the seven compounds was
investigated in vitro on HepG2, MCF-7, Bel-7402, HeLa,
and A549 cells (Table 1). The well-known anticancer
chemotherapy drug 5-fluorouracil (5-FU) was used as a
control. Antitumor activity was observed for all the
compounds, except for A2 and A3, which showed no
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8
Anti-Cancer Drugs 2018, Vol 00 No 00
Fig. 5
(a)
*
CICCP
A7
12
10
8
*
*
*
6
4
2
0
25.0
Control
12.5
Concentration (μg·mL-1)
(b)
100
80
*
*
60
6.25 μg·mL-1 A7
Control
40
20
0
Control 6.25
12.5
25.0
12.5 μg·mL-1 A7
25.0 μg·mL-1 A7
Concentration of A7 (μg·mL-1)
Rh123
Disturbance in intracellular Ca2 + concentration and mitochondrial membrane potential (Δψ). (a) Fluo-3 AM staining for the level of intracellular Ca2 +
concentration. Data are presented as mean SD (N =3).*P < 0.01 versus the control. (b) Rh123 staining for changes in the mitochondrial membrane
potential (Δψ). The left panel shows the fluorescence histograms of a representative test. Data are presented as mean SD in the right panel.
*P < 0.05 versus the control. The mean fluorescence for control cells was artificially set as 100.
effect on either of the five tumor cell lines. These two
compounds lack a methyl group in the amido of Bn-Leu.
These results suggested that the methyl-N in leucine is
crucial for the antitumor activity of these leucine dipep-
tides, which is consistent with a previous report on
compounds with methyl-N have antitumor activity [10].
In addition, we also found that when one of the protec-
tion groups (Boc or Bn) was removed, all of the com-
pounds lost their antiproliferation activity (data not
shown). A6 effectively inhibited the proliferation of all
five cell lines, with an IC50 ranging from 8.09 to 15.98 μg/
ml, which is comparable to that of 5-FU. Compounds A1,
A4, A5, and A7 were selectively active against HepG2,
MCF-7, and Bel-7402. These five compounds are dif-
ferent D/L stereomers and all of them have a methyl
group in the amido of Bn-Leu. Among all the compounds
tested, A7, the only stereomer condensed by double D-
Leu, showed the highest antitumor cell activity, which
was comparable to that of galaxamide. Therefore, this
compound was selected for more detailed studies.
of exposure, which showed that A7 had a significant
inhibitory effect on cell growth and viability in a
time-dependent and concentration-dependent manner
(Fig. 2). The IC50 values were 19.35 μg/ml (24 h), 11.08
μg/ml (48 h), and 4.78 μg/ml (72 h), respectively. At the
72 h time-point, the IC50 values of 5-FU and etoposide
(Eto), two traditional anticancer drugs that were used as
controls, were 29.43 and 18.01 μg/ml, respectively. Thus,
A7 is a promising antitumor compound, and its strong
tumor-inhibitory effect led us to study its potential
mechanism of action.
A7 inhibits cancer cells by cell cycle arrest and apoptosis
induction
To unravel the potential mechanisms of the tumor-
inhibitory effect of A7, HepG2 cells were treated with
6.25, 12.5, and 25.0 μg/ml of A7 for 24 h and subjected to
flow cytometry analysis for cell cycle distribution
(Fig. 3a). Cells that arrested in the S phase increased from
19.9 3.3 to 26.4 2.5, 30.5 2.9, and 37.5 3.1%,
respectively. This was associated with a concomitant
decrease in the percentage of G1 cells. The fraction of
cells in the sub-G1 phase, a feature of nuclear apoptosis,
We tested the in-vitro activity of A7 on HepG2 cells
with a wider dose range and various exposure durations
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Antineoplastic activity of Leu dipeptide Lei et al.
9
also increased from 3.5 1.1 to 6.5 2.5, 15.3 1.4, and
21.3 2.1% for the three doses of A7, respectively. In
addition, the population of Annexin-V positive/propi-
dium iodide-negative cells increased significantly in a
time-dependent and dose-dependent manner, which
suggested the induction of early apoptosis (Fig. 3b).
Morphological observation detected marked changes that
are typical to cells undergoing apoptosis, including loss of
focal adhesion (Fig. 3c, panel a), nuclear condensation
(Fig. 3c, panel b), margination, aggregation, and con-
densation of nuclear chromatin (Fig. 3c, panel c). When
observed under an atomic force microscope, untreated
cells showed features common to cancer cells, with an
average cell height of 2.72 0.09 μm and a cell diameter
of 12.30 0.21 μm (Fig. 3d). Cells cultured in the pre-
sence of A7 for 24 h showed a dose-dependent decrease
in cell height together with an enlarged cell diameter.
These findings indicated that A7 induced apoptotic
volume decrease, which is an early prerequisite to
apoptosis. Taken together, these results suggest that A7
inhibited the proliferation of cancer cells by inducing cell
cycle arrest and apoptosis.
design [11]. Although the cyclic penta-leucine-peptide,
galaxamide that exhibited potent antineoplastic activity was
successfully synthesized in our previous study, we are still
challenged by the low yield and poor purity of galaxamide.
In this study, we designed and synthesized a series of
leucine homo-oligopeptides and identified compounds that
were biologically active against tumors. So far, oligopep-
tides that have shown biological activities are almost all
hetero-oligomers, and the shortest anticancer peptide
compound in clinical trials is a tripeptide with an aromatic
amino-acid residue (Tyr) with a functional benzene ring
[12]. Here, we have shown that homo-oligopetides of ali-
phatic and nonpolar amino acids were also bioactive against
tumor cells. In addition, one leucine homodipeptide, A7,
showed antineoplastic effects comparable to 5-FU and
galaxamide. To our knowledge, A7 is the shortest homo-
oligopeptide, and this is the first report that a linear homo-
oligopeptide showed strong antitumor activity through
apoptosis induction.
Generally, chemical modification of primary peptides has
been used to improve the efficiency and therapeutic
potential. One of the most frequently used strategies for
such modification is to replace amino groups with other
functional groups [13]. Another typical method is to
incorporate a side-chain-modified amino-acid residue or
other amino-acid analogs, such as D-amino acid, β-amino
acid, and N-methylated amino acid [14]. Introduction of
these modified amino acids into natural peptides usually
exerts noticeable effects in their biological activities.
Moreover, modification of marine cyclic peptides using
special amino acid and nonamino acid fragments has
been found to highly improve their bioactivities and
makes them better lead compounds for potential appli-
cation [15]. In our study, substitution of Bn-Leu with an
N-methylated one was crucial for the antineoplastic
activity of the dipeptide product. Dipeptide derivatives
without this modification (A2 and A3) showed no inhi-
bitory effects on tumor cells (Table 1). This might
explain the widest antitumor spectrum of A6, which was
condensed with two N-methylated leucines. In addition,
D/L stereochemistry also affected the antitumor proper-
ties of the leucine dipeptide derivatives; D-leucine was
beneficial over L-leucine. This might be because of the
fact that human cells typically utilize L-amino acids for
protein synthesis, and D-amino acids are produced by
post-translational modification of proteins, which are
found in bacteria and some marine organisms [16]. Thus,
it is conceivable that replacing the two L-leucines in A6
with D-leucines will generate a new dipeptide with
higher antitumor activity.
A7 induced cleavage of caspase-9 and caspase-3 in
HepG2 cells
After treatment of HepG2 cells with different concentra-
tions for 24 h, A7 upregulated levels of cleaved caspase-9
and caspase-3 in a concentration-dependent manner
(Fig. 4a). Treatment of HepG2 cells with 6.25 μg/ml of A7
was sufficient to induce the cleavage of caspase-9. The
amount of cleaved caspase-9 increased gradually with
increasing A7 concentration. A similar effect of A7 was also
observed on caspase-3. Colorimetric assay also detected a
dose-dependent increase in the total level of caspase-3
upon A7 treatment, but no change was found for caspase-8
(Fig. 4b). These results support the idea that A7 acts as an
antitumor agent by inducing cell apoptosis.
A7 alters intracellular Ca2 + concentrations and
mitochondrial membrane potential (Δψ)
HepG2 cells were treated with an increasing dose of A7 for
24 h, followed by cytoflurometric analysis to study Ca2 +
release and Δψ. Exposing the cells to 12.5 and 25.0 μg/ml
of A7 increased the intracellular Ca2 + level by more than
four times, and the same tendency was also observed
for cells treated with cyanide-m-chlorophenylhydrazone
(Fig. 5a). This was accompanied by a dose-dependent
decrease in Δψ (Fig. 5b), which indicated that A7
increased intracellular Ca2 + levels and decreased the
mitochondrial membrane potential. This result served as
evidence of the effect of A7 on apoptosis induction.
Cancer cells usually show an elevated threshold for
apoptosis and defects in apoptosis pathways. Peptides
that can induce apoptosis in cancer cells are considered
prime candidates for the development of novel antitumor
drugs. Many naturally derived peptides have been pro-
ven to exert their antiproliferative activity through
Discussion
Naturally cyclic peptides are exceptionally resistant to
denaturing and digestion. They function mainly as toxins
or antimicrobial substances in vitro and have been used
as scaffolds, precursors, and lead compounds for drug
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10 Anti-Cancer Drugs 2018, Vol 00 No 00
apoptosis induction [17]. In our study, in the presence of
A7, atomic force microscopy observed apoptotic volume
decrease, which is considered to be an early prerequisite
to apoptosis that precedes key biochemical hallmarks of
apoptosis. The apoptosis-induction effect of A7 was
validated by several approaches commonly used to
study cells undergoing apoptosis. The superior tumor-
inhibitory effect of A7 over traditional chemotherapy
drugs (e.g. 5-FU and Eto) suggests that it may be a
promising candidate for future application. The obvious
selectivity of leucine dipeptides against different cell
lines suggested the involvement of cell type-dependent
mechanisms in their activity.
and 41506156), the Natural Science Foundation of
Guangdong Province (2016A030310095), the Guangdong
Provincial Science and Tech Project (2015A020211016),
and the Guangzhou Industry-Academia-Research Colla-
borative Innovation Project (201604016009).
Conflicts of interest
There are no conflicts of interest.
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Acknowledgements
The authors thank Dr. Jun Fan of Institute for Virus
Research of Kyoto University for her critical review of this
manuscript. This work was supported by grants from the
National Natural Science Foundation of China (21672084
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