Biomedical Research (Tokyo) 37 (2) 153–159, 2016
Combination of aspartic acid and glutamic acid inhibits tumor cell proliferation
Yoshie YAMAGUCHI, Katsunori YAMAMOTO, Yoshinori SATO, Shinjiro INOUE, Tetsuo MORINAGA, and Eiichi
H
IRANO
Department of Placenta avenir research institute, Japan Bio Products Co., Ltd., 1488-4 Fukuoka Bio Factory 201, Aikawa, Kurume,
Fukuoka, 839-0861, Japan
(Received 10 February 2016; and accepted 15 February 2016)
ABSTRACT
Placental extract contains several biologically active compounds, and pharmacological induction
of placental extract has therapeutic effects, such as improving liver function in patients with hepa-
titis or cirrhosis. Here, we searched for novel molecules with an anti-tumor activity in placental
extracts. Active molecules were separated by chromatographic analysis, and their antiproliferative
activities were determined by a colorimetric assay. We identied aspartic acid and glutamic acid
to possess the antiproliferative activity against human hepatoma cells. Furthermore, we showed
that the combination of aspartic acid and glutamic acid exhibited enhanced antiproliferative activi-
ty, and inhibited Akt phosphorylation. We also examined in vivo tumor inhibition activity using
the rabbit VX2 liver tumor model. The treatment mixture (emulsion of the amino acids with Lipi-
odol) administered by hepatic artery injection inhibited tumor cell growth of the rabbit VX2 liver.
These results suggest that the combination of aspartic acid and glutamic acid may be useful for
induction of tumor cell death, and has the potential for clinical use as a cancer therapeutic agent.
Hepatocellular carcinoma (HCC) is the sixth most
common and aggressive malignancy in the world
(22). Poor prognosis in HCC is the major cause of
cancer-related deaths, and each year more than
500,000 new patients are diagnosed with HCC
worldwide (4). Furthermore, increasing incidence
has been projected through 2020 (22). Various types
of therapies are available for HCC such as transarte-
rial therapy with or without embolization, systematic
therapy, interferon, and lamivudine. Although con-
ventional chemotherapy is well tolerated in inopera-
ble HCC patients (27, 32), various adverse effects
have been reported for interferon (8). In addition,
resistance to chemotherapy due to long-term admin-
istration of anticancer drugs is widely identied in
HCC patients; the response rates are approximately
20% for single agent as well as combination chemo-
therapy (29). In addition, the 3-year tumor recurrence
rate was under 60% following hepatic resection (19)
and the 5-year overall survival rate ranged between
39% and 50% (14, 23). Therefore, cure rates are not
satisfactory (2).
 
Placental extract can be obtained by hydrolysis of
placenta via both hydrochloric acid and enzymatic
digestion. It has several biological activities, such as
stimulation of liver regeneration (28), anti-oxidation
(26), and anti-xanthine oxidase activity (30). Owing
to these multiple functions, placental extract is ex-
pected to have wide applications in the healthcare
eld. In fact, human placental extract has been clini-
cally administered to improve liver function in pa-
tients with hepatitis or cirrhosis in Japan for more
than 50 years. Based on the cell growth-promoting
activity of placental extract (1, 28), we hypothesized
that placental extract may be a useful substitute for
fetal bovine serum in cell culture systems. However,
contrary to our expectations, placental extract inhib-
Address correspondence to: Eiichi Hirano, Ph.D
Department of Placenta avenir research institute, Japan
Bio Products Co., Ltd., 1488-4 Fukuoka Bio Factory
201, Aikawa, Kurume, Fukuoka, 839-0861, Japan
Tel: +81-9-4234-2216, Fax: +81-9-4234-2333
E-mail: ehirano@placenta-jbp.co.jp
Y. Yamaguchi et al.154
USA). The active fractions of the placental extract
were subjected to ion exchange chromatography
SP-sepharose (Amersham Pharmacia Biotech, Pisca-
taway, NJ, USA), and then applied to a high-perfor-
mance liquid chromatography (HPLC) uRPC C2/
C18 ST4.6/100 column (Amersham Pharmacia Bio-
tech, Piscataway, NJ, USA). This procedure was re-
peated twice. The separated active components were
analyzed by postcolumn derivatization with O-
phthaldialdehyde using HPLC.
Western blot analysis. Cells were plated at 8.0 × 10
5
cells/60-mm dish and allowed to grow overnight.
Then cells were treated with compounds in serum
containing DMEM medium for 2 h. After treatment,
cells were lysed in cell lysis buffer containing prote-
ase and phosphatase inhibitors. Cell lysates (20 μg
protein) were loaded onto 10% SDS-PAGE gels,
electrophoresed under reducing conditions, and trans-
ferred onto PVDF membranes. Blots were probed
with anti-phospho-p44/42 MAPK (Erk1/2; Thr202/
Tyr204; D13.14.4E; Cell Signaling Technology, Inc.,
Danvers, MA, USA), anti-p44/42 MAPK (Erk1/2;
137F5; Cell Signaling Technology, Inc.), anti-AKT
(AKT; pan; C6707E; Cell Signaling Technology,
Inc.) and anti-phospho-AKT (Phospho-AKT; Ser473
D9E; Cell Signaling Technology, Inc.) antibodies,
and horseradish peroxidase (HRP)-conjugated sec-
ondary antibodies (Cell Signaling Technology, Inc.).
Following washes, the membranes were developed
with WesternSure premium chemiluminescent sub-
strate (LI-COR; Lincoln, NE, USA) and read on a
LAS-3000 LuminoImage analyzer (Fujilm, Tokyo,
Japan).
In vivo studies using rabbit VX2 liver tumors. The
aqueous mixture solution of aspartic acid (4.0 mg/
mL) and glutamic acid (4.4 mg/mL) containing 1.53%
Tween-20 was mixed with 5.5 volumes of Lipiodol
(Terumo, Japan), and stirred with an ultrasonic stirrer.
VX2 carcinoma cells were maintained as a tumor
line in SLC Biotechnical Center. Adult JW/CSK
male rabbits weighing 2.6–3.1 kg (SLC, Shizuoka,
Japan) underwent VX2 tumor implantation to the
left medial hepatic lobe. The animals were used for
experiments two weeks after tumor implantation.
The tumors were measured with Vernier calipers,
and the tumor sizes at the time of the experiment
were between 1.0 and 2.0 cm in diameter. In all ex-
periments, anesthesia was administered by intramus-
cular injection of a mixture of ketamine hydrochloride
(1.1 mL/kg, Daiichi-Sankyo, Japan) and xylazine
hydrochloride (0.4 mL/kg, Bayel, Japan). Eighteen
ited growth of various cell lines such as B16 (mouse
skin melanoma) and HepG2 (human hepatoma) cells,
but not primary cells such as rat hepatocytes. This
finding suggested that placental extract contains
compounds with antiproliferative activity. Hence, we
searched for novel compounds with anti-cancer ac-
tivity in placental extract using cell-based assays.
We used HepG2 cells for the following two reasons:
placental extract has been used in clinical practice
for treatment of hepatic diseases, and our pilot cell
growth assay showed that placental extract inhibited
the proliferation of these cells.
 
In the present study, we identified compounds
with antiproliferative activity in placental extract us-
ing chromatographic analyses, and determined the
optimal combination of these compounds for effec-
tiveness. The antiproliferative effects of these com-
pounds on HCC tumor cells were evaluated in
HepG2 cells. To determine the signaling pathways
involved in the antiproliferative activity, we exam-
ined kinase phosphorylation associated with cell
growth or survival. Furthermore, we evaluated the
efciency of these compounds in vivo in a conven-
tional animal tumor model.
MATERIALS AND METHODS
Chemicals. Most chemicals were purchased from
Wako Chemicals (Tokyo, Japan) and Nacalai Tesque
(Kyoto, Japan).
Cell line and cell culture. The placental extract was
obtained from Japan Bio Products Co., Ltd. (Tokyo,
Japan). HepG2 (hepatoma), Huh-7 (hepatocellular
carcinoma), and HLE (hepatoma) cells were pur-
chased from Health Science Research Resources
Bank (Osaka, Japan). The cells were cultured in Dul-
becco’s modied Eagle medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS), 2 mM
L-glutamine, 100 U/mL penicillin, and 100 μg/mL
streptomycin at 37°C in humidied air containing
5% CO
2
.
Cell viability assay. The cells were plated at 1.5 ×
10
3
cells/well in 96-well plates, and cultured for
24 h. On the following day, the test samples were
added, and the cells were cultured for an additional
hour. Cell viability was determined by sulforhoda-
mine B.
Purication and identication of active components.
The placental extract was separated by Tangential
Flow Filtration system (Millipore, Bedford, MA,
The effect of amino acids in tumor cell proliferation 155
mixtures lacking Ala, Gly, Ser, or Thr alone showed
almost equivalent cell growth inhibition activity rel-
ative to the Master solution consisting of the six
amino acids mentioned above. In contrast, mixtures
lacking Asp and Glu showed lower cell growth inhi-
bition activity. Hence, Asp and Glu are the responsi-
ble components for the inhibition of HepG2 cell
growth. Moreover, as we did not observe cell growth
inhibition activity in mixtures lacking Asp or Glu
alone (Fig. 1C), we evaluated various combinations
of amount and treatment duration to nd the most
effective one. We found that the combination of
3 mM Asp with 3 mM Glu showed the highest inhi-
bition activity (Fig. 1D).
Effect of the combination of Asp and Glu on various
liver tumor cell lines
Furthermore, treatment with the combination of
6 mM Asp with 6 mM Glu for 24 h greatly reduced
viability of HepG2, Huh-7, and HLE cells (38, 47,
and 39%, respectively) (Fig. 2). Treatment for 48
and 72 h could further reduce cell viability below
20% (Fig. 2). Especially, the viability of HLE cells
treated for 72 h was below 10%. These results indi-
cated that the combination of Asp and Glu could in-
hibit growth of liver tumor cell lines at a dose- and
time-dependent manner.
The combination of Asp and Glu inhibits Akt signal-
ing pathway in HCC cell line
We hypothesized that the combination of Asp and
Glu is required to regulate activities of specic pro-
tein kinases in signaling pathways essential for ma-
lignant cell growth, such as the Raf/MEK/ERK and
PI3K/PTEN/Akt/mTOR pathways (12, 13, 16–17,
25). Therefore, we examined the phosphorylation of
ERK and Akt in Huh-7 cells treated with Asp and
Glu using western blot. In these cells, the total levels
of ERK and Akt and the level of ERK phosphoryla-
tion were unchanged, whereas the level of phospho-
Akt was greatly decreased, relative to the control
(Fig. 3). The selective MEK inhibitor Sorafenib or
phosphoinositide 3-kinases inhibitor LY294002 used
as a control, inhibited ERK or Akt phosphorylation
at 10 μM or 50 μM, respectively, which are consist-
ed with the induction of a feedback loop upon the
inhibition of phosphor-ERK or -Akt signaling in this
cell.
Effect of the combination of Asp and Glu in the rab-
bit VX2 liver tumor model
To determine the antitumor activity of the combina-
tion of Asp and Glu in vivo, we used the rabbit
VX2 tumor-bearing rabbits were randomly divided
into two groups (emulsion treatment group and
without emulsion group), and hepatic artery injec-
tion was performed in these animals (15). The emul-
sion treatment group received saline (0.1 mL/kg) or
aspartic acid (0.7 or 1.4 mg/kg) and glutamic acid
(0.8 or 1.5 mg/kg) in 0.01% Tween-20 mixed with
Lipiodol. The without emulsion group received as-
partic acid (0.7 or 1.4 mg/kg) and glutamic acid (0.8
or 1.5 mg/kg) in 0.01% Tween-20. Tumor dimen-
sions and body weights were measured 7 days after
drug administration. Tumor volumes were calculated
using the equation (l × w
2
) / 2, where l and w refer
to the larger and smaller dimensions collected at
each measurement. Hepatic and renal toxicities were
determined by biochemical analysis of the plasma of
non-tumor bearing rabbits. Blood samples were col-
lected before and 1, 3, 7, and 14 days after drug ad-
ministration.
Ethics Statement. Animal experiments were carried
out in accordance with the protocol approved by the
Animal Care and Use Committee and in compliance
with SLC guidelines. Animals received food and
water ad libitum.
Statistics. The in vitro and in vivo data are presented
as mean ± SD from at least three replicates and were
analyzed by Dunnett’s test. A p-value < 0.05 was
considered signicant.
RESULTS
Identication of active components in the placental
extract
To determine whether the placental extract has tumor
cell growth inhibition activity, we treated HepG2
cells with the placental extract and evaluated cell
growth by sulforhodamine B assay. As shown in
Fig. 1A, the placental extract inhibited the prolifera-
tion of HepG2 cells dose-dependently. Next, to iden-
tify the main antiproliferative components of the
placental extract, we analyzed the composition of
the placental extract using chromatographic methods
(Fig. 1B). We performed thin-layer chromatography
and HPLC (O-phthaldialdehyde-derivatization) anal-
ysis, and our data suggested that the nal fraction
contained six of amino acids including alanine (Ala),
aspartic acid (Asp), glutamic acid (Glu), glycine
(Gly), serine (Ser), and threonine (Thr) (data not
shown). To determine which amino acids are re-
sponsible for the inhibition of HepG2 cell growth,
we performed a deletion assay. As shown in Fig. 1C,
Y. Yamaguchi et al.156
DISCUSSION
In this study, we found that human placental extract
suppresses the growth of human hepatocellular cells,
and identied the active components to be Asp and
Glu. The combination of these substances showed
antiproliferative activity in a wide variety of tumor
cell lines (data not shown). As deletion of Asp and
Glu reduced the antiproliferative activity in the nal
fraction from chromatography, we concluded that
these amino acids were the active components in the
placental extract. However, since the deletion of
these amino acids did not cause a complete loss of
antiproliferative activity, we did not exclude the
possibility of the presence of other active substances
VX2 liver tumor model. Lipiodol was used as a
drug carrier agent to effectively induce the activity
of Asp and Glu in the animals. The body weights of
VX2 rabbits did not change signicantly in the ex-
perimental groups (data not shown). Growth ratios
of the liver tumors were determined at Day 7 after
hepatic artery injection. The growth ratio of tumors
in the low-dose Asp and Glu with Lipiodol group
was −26.3 ± 31.7, and −33.9 ± 13.0 in the high-dose
group (Table 1). There was a signicant difference
between the ratio of the treatment group and that of
the control group. However, administration of high-
dose Asp or Glu without Lipiodol showed no effect
on tumor inhibition (Table 1).
Fig. 1
 
Identification of anti-proliferative active components in the placental extract. Effect of the placental extract on the
growth of hepatoma cells. HepG2 cells were incubated with serial dose of the placental extract or PBS (as a control) for
48 h and analyzed using the sulforhodamine B method. Data are presented as mean ± SD (n = 3) from 3 independent ex-
periments (A). Chromatographic partial purification of active components in the placental extract. Summary of chromato-
graphic methods for partial purification of active components (B). Single amino acid deletion assay to determine the
components with antiproliferative activity. Amino acid mixtures with single amino acid deletion or PBS (as a control) or Mas-
ter (containing Ala, Asp, Glu, Gly, Ser, or Thr alone) were added to HepG2 cells and cultured for 48 h in DMEM containing
10% FBS. Data are presented as mean ± SD (n = 3) from 2 independent experiments (C). Screening high potency combi-
nation of antiproliferative activity among amino acids. Combinatorial antiproliferative activity of Asp and Glu was observed.
HepG2 cells were incubated with serial dose of Asp or Glu for 48 h and subjected to sulforhodamine B assay. Test samples
were added to HepG2 cells and cultured for 48 h in DMEM containing 10% FBS. Data are presented as mean ± SD (n = 3)
from 2 independent experiments (D).
The effect of amino acids in tumor cell proliferation 157
in the nal fraction. In fact, we observed peaks at
254 and 280 nm in the HPLC analysis of the nal
fraction (data not shown), which suggested the exis-
tence of molecules with an aromatic group and a
nucleobase.
 
Amino acids function as substrates for protein
synthesis, are metabolized as an energy source for
protein production, and modulate numerous cellular
functions (6). In addition, a number of studies have
reported the anti-tumor activity of free amino acids.
For example, one branched-chain amino acid (BCAA)
could directly suppress HepG2 cell growth (9). Al-
though isoleucine did not show any antiproliferative
activity against colon cancer cells, it prevented tu-
mor metastasis (24). Aspartic acid and glutamic acid
are known mainly to act as neurotransmitters, and
have not been reported to have tumor inhibition ac-
tivity (3, 11). In our observation, although Asp or
Glu alone only showed slight antiproliferative activ-
ity, their combination showed a 36% increased anti-
proliferative activity. These results suggested that
Asp and Glu have synergistic effects on tumor
suppression. Recent studies indicated that a func-
tional N-methyl-D-aspartate receptor (NMDAR) was
expressed in HepG2 and Huh-7 cells (20), and
NMDAR-dependent signaling would be related with
cancer cell growth (10, 18, 31). Therefore, Asp and
Glu may function through NMDAR to inhibit tumor
cell proliferation.
 
In our experiments, we observed that the combi-
nation of Asp and Glu inhibited Akt phosphorylation
in Huh-7 cells. These results indicated that tumor
cell death induced by Asp and Glu is likely through
the Akt pathway. Akt belongs to the serine/threonine
kinase family, and plays a crucial role in the regula-
tory network of the cell and affects virtually all cel-
lular activities, especially the growth and survival of
tumor cells (16–17). The PI3K/Akt/mTOR signaling
pathway plays a central role in the regulation of cell
Fig. 3
 
The combination of Asp and Glu inhibits Akt path-
way in liver tumor cells. Huh-7 cells were treated with 3 mM
Asp and 3 mM Glu in DMEM containing 10% FBS for 2 h.
Cells were lysed and 20 μg of protein was used for SDS-
PAGE. Protein phosphorylation was detected by Western
blot analysis.
Fig. 2
 
Antiproliferative activity of the combination of Asp and Glu in liver tumor cell lines. The combination of Asp and Glu
were added to HepG2, Huh-7, and HLE cells and cultured for 24 to 72 h in DMEM containing 10% FBS. Treatment with the
combination of Asp and Glu for 24 h (open circle), 48 h (closed circle), and 72 h (open triangle). Data are presented as
mean ± SD (n = 3) from 3 independent experiments.
Y. Yamaguchi et al.158
plication using this method, which would increase
its clinical benets.
 
In conclusion, we identified and optimized the
combination of Asp and Glu as a potent tumor cell
growth inhibitor from the placental extract. The
combination of Asp and Glu predominantly induced
necrotic cell death in HCC cell lines in a dose- and
time-dependent manner, and its mode of action
would be inhibition of Akt phosphorylation. Further-
more, the combination of Asp with Glu efcacy was
also demonstrated in vivo using the rabbit VX2 liver
tumor model. These results suggest that the combi-
nation of Asp and Glu may be used for induction of
tumor cell death and could have clinical application
as a cancer therapeutic agent.
Acknowledgement
We thank Ms. Asami Sugimoto (Biotechnical Cen-
ter, Japan SLC, Inc.) for helping in vivo study.
REFERENCES
proliferation, migration, survival and angiogenesis,
and is often dysregulated in HCC (12, 25), making
it an attractive target for anticancer therapy. Indeed,
a previous study reported that Akt phosphorylation
level was up-regulated in HCC (13). In addition,
perifosine, an Akt specic inhibitor, had been tested
in phase II clinical trials (21). Therefore, with the
inhibition activity on Akt phosphorylation, the com-
bination of Asp and Glu may be a promising cancer
therapy agent.
 
The combination of Asp and Glu exhibited syner-
gistic effect on antiproliferative activity against tu-
mor cells. Herein, the arterial chemoembolization
method seems to be extremely helpful as a way of
utilizing the combination of Asp with Glu in vivo.
Iodized oil (Lipiodol) has been used as an embolic
agent and a carrier of anticancer drugs. It is capable
of selective accumulation and retained for a long
period in hypervascular hepatic tumors. In addition,
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and Lipiodol were mixed manually to prepare an
oil-in-water emulsion. These mixtures tended to sep-
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addition, a recent study reported greater inhibition
of tumor angiogenesis in rabbits with VX2 cancer
after arterial heated Lipiodol infusion compared to
Lipiodol infusion (5). Since the combination of Asp
and Glu is quite thermostable, it is suitable for ap-
Table 1
 
Effect of the combination of Asp and Glu against rabbit VX2 liver tumors
Test samples Mean tumor volume (cm
3
) Mean tumor growth ratio (%)
Before administration At experimental period
Control (saline) 0.66 ± 0.31 3.16 ± 2.08 355.3 ± 90.1
Lipiodol only 0.68 ± 0.44 1.84 ± 0.96 263.9 ± 344.5
Low-dose Asp and Glu (0.7 mg/kg Asp
and 0.8 mg/kg Glu) without Lipiodol
0.42 ± 0.11 1.92 ± 1.90 309.5 ± 313.2
High-dose Asp and Glu (1.4 mg/kg Asp
and 1.5 mg/kg Glu) without Lipiodol
0.36 ± 0.16 1.94 ± 1.16 445.4 ± 167.9
Low-dose Asp and Glu (0.7 mg/kg Asp
and 0.8 mg/kg Glu) with Lipiodol
0.84 ± 0.30 0.67 ± 0.50 −26.3 ± 31.7*
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and 1.5 mg/kg Glu) with Lipiodol
0.53 ± 0.11 0.34 ± 0.02 −33.9 ± 13.0*
Data are presented as mean ± SE (n = 3). *Signicant difference (P < 0.05) between tumor size (in two perpendicular dimen-
sions) and treatment with test samples or control (n = 3 per group).
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