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Chemico-Biological Interactions 223 (2014) 19

Contents lists available at ScienceDirect

Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Astin B, a cyclic pentapeptide from Aster tataricus, induces apoptosis


and autophagy in human hepatic L-02 cells
Li Wang, Ming-Dan Li, Pei-Pei Cao, Chao-Feng Zhang , Fang Huang, Xiang-Hong Xu,
Bao-Lin Liu, Mian Zhang
Research Department of Pharmacognosy, China Pharmaceutical University, Longmian Road 639, Nanjing 211198, PR China

a r t i c l e

i n f o

Article history:
Received 15 February 2014
Received in revised form 21 August 2014
Accepted 4 September 2014
Available online 16 September 2014
Keywords:
Astin B
Liver injury
Apoptosis
Autophagy
Aster tataricus

a b s t r a c t
Astins (including astin B) are a class of halogenated cyclic pentapeptides isolated from the medicinal herb
of Aster tataricus. However, our previous works showed that the herbal medicine was hepatotoxic in vivo,
and a toxicity-guided isolation method led to the identication of a cyclopeptide astin B. Astin B is structurally similar to cyclochlorotine, a well-known hepatotoxic mycotoxin. Thus, the aim of this study was
to determine the potential cytotoxic effects and the underlying mechanism of astin B on human normal
liver L-02 cells. We found that astin B has hepatotoxic effects in vitro and in vivo and that hepatic injury
was primarily mediated by apoptosis in a mitochondria/caspase-dependent manner. Astin B provoked
oxidative stress-associated inammation in hepatocytes as evidenced by increased levels of reactive oxygen species (ROS), reduced contents of intracellular glutathione (GSH), and enhanced phosphorylation of
c-Jun N-terminal kinase (JNK). Furthermore, the mitochondria-dependent apoptosis was evidenced by
the depolarization of the mitochondrial membrane potential, the release of cytochrome c into cytosol,
the increased ratio of Bax/Bcl-2, and the increased activities of caspases-9 and -3. Interestingly, astin B
treatment also induces autophagy in L-02 cells, characterized by acidic-vesicle uorescence, increased
LC3-II and decreased p62 expression. Autophagy is a protective mechanism that is used to protect cells
from apoptosis. The presence of autophagy is further supported by the increased cytotoxicity and the
enhanced cleaved caspase-3 after co-treatment of cells with an autophagy inhibitor, also by increased
LC3-II and decreased p62 after co-treatment with a caspase inhibitor. Taken together, astin B, most likely
together with other members of astins, is the substance that is primarily responsible for the hepatotoxicity of A. tataricus.
2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
The hepatocyte is especially vulnerable to injury due to its central role in xenobiotic metabolism including the metabolism of
drugs. Therefore, drug-induced liver injury is the most frequent
reason for post-marketing warnings and withdrawal [1]. The death
of hepatocytes and other types of hepatic cells is a characteristic
feature of drug-induced injury [2]. Based on morphological appearance, cell death has been classied into several modes such as
apoptosis, necrosis, necroptosis, autophagy, and cornication [3].
Of these, apoptosis is considered to be a common pathway for
execution of hepatocytes upon liver injury. In response to xenobiotic metabolism, hepatocytes often generate excess reactive
oxygen species (ROS), which evoke oxidative stress-associated
Corresponding authors. Tel./fax: +86 25 86185137.
E-mail addresses: njchaofeng@126.com (C.-F. Zhang), mianzhang@126.com
(M. Zhang).
http://dx.doi.org/10.1016/j.cbi.2014.09.003
0009-2797/ 2014 Elsevier Ireland Ltd. All rights reserved.

inammation, leading to mitochondrial dysfunction [4,5].


Mitochondria play a key role in the regulation of redox homeostasis and apoptosis in cells [6]. A loss of mitochondrial function
allows the release of a number of proapoptotic factors, such as
cytochrome c, which induces caspase activation to trigger
mitochondria-dependent apoptotic cell death [7]. Accumulating
evidence demonstrates that hepatocyte apoptosis is tightly associated with drug-induced liver injury [6,8]. However, autophagy is
understood to be a mechanism of protection against various forms
of human diseases, including drug-induced liver injury, with an
extremely complex interplay [9,10].
Astins, mainly including astins AI, are a class of natural halogenated cyclic pentapeptides isolated from the root of Aster tataricus
L. f. (RAT, Compositae) that has been used for over 2000 years in
traditional Chinese medicine for the relief of coughs and the
removal of phlegm. This class of compounds exhibits antitumor
and immunosuppressive activities [1113]. However, our previous
studies discovered the potent hepatotoxicity of RAT in mice, and a

L. Wang et al. / Chemico-Biological Interactions 223 (2014) 19


Cl

HN

Cl
O
NH

HN

OH
N
H

OH

180

Relative viability

0.9

0.8
0.7

*
*

140

0.6

0.5

160

0.4
0.3

LDH release (%)

1.1

120
100
80
60
40
20
0

15

30

45

60

15

Astin B (M)

30

45

60

Astin B (M)

Fig. 1. Effects of astin B on cell viability and lactate dehydrogenase (LDH) activity in L-02 cells. (A) Chemical structure of astin B. (B) Cells were treated with astin B for 12, 24,
and 48 h, and cell viability was determined by the MTT assay. (C) Cells were treated with astin B for 24 h, and LDH leakage was measured using commercially available kits.
Data in B and C are expressed as the mean SD from three independent experiments with n = 6 for MTT and n = 4 for LDH. p < 0.05 compared with the control group (0 lM).

toxicity-guided isolation method led to the identication of an


astin-rich fraction (71.2% of the relative content), from which a
halogenated cyclic pentapeptide astin B (Fig. 1A) was given [14].
Astin B is structurally similar to cyclochlorotine, a well-known
hepatotoxic mycotoxin isolated from Penicillium islandicum [15],
but the hepatotoxic effects and the underlying mechanisms were
unknown. In this study, we evaluated the effects of astin B on
the modulation of cell death, apoptosis and autophagy in human
normal liver L-02 cells. Our experiments indicate that astin B has
marked toxic effects in vitro and in vivo and induces hepatic cell
death mainly by apoptosis through a mitochondria/caspasedependent pathway. Astin B also induces autophagy in L-02 cells,
which appears to protect cells from apoptosis to some extent.
2. Materials and methods
2.1. Drugs and chemicals
Astin B (Fig. 1A) was obtained from previous experiments [14]
with a purity of more than 98%. Chlorogenic acid (CGA) was
obtained from Nanjing Zelang Medical Technology Company with
a purity of 98% (Nanjing, China). For experiments in cells, solutions
of astin B and CGA were prepared in DMSO and diluted to the
desired concentrations in FBS-free medium. The DMSO concentrations in the experiments never exceeded 0.1%.
Fetal bovine serum (FBS) was obtained from Hyclone (Thermo,
South America). RPMI-1640 medium was obtained from Gibco
BRL (NY, USA.). Earles balanced salts solution (EBSS), 3-methyladenine (3-MA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from SigmaAldrich (St.
Louis, USA.). Antibodies for c-Jun N-terminal kinase (JNK), phosphorylated JNK (pJNK), Bax, Bcl-2, and caspase-3 were purchased
from Cell Signal Technology Inc. (Massachusetts, USA). Anti-LC3
antibody was obtained from MBL (Nagano, Japan). Anti-b-actin
antibody and secondary anti-mouse and anti-rabbit antibodies

were obtained from Lianke Biotechnology (Hangzhou, China). All


of the other reagents were purchased from Amresco (Ohio, USA).
2.2. Cell culture
L-02 cells, a normal human liver cell line from the Cell Bank of
the Chinese Academy of Sciences (Shanghai, China), were grown in
RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin and 100 lg/mL streptomycin and incubated at 37 C in a
humidied atmosphere (5% CO2). The medium was renewed every
2 days until the cells were grown to conuence.
2.3. Cytotoxicity assay
L-02 cells were seeded at an initial density of 1  105 cells/mL
in 96-well plates for 24 h and were incubated with fresh medium
containing different concentrations of astin B for 12, 24, and
48 h. After incubation, MTT was added to each well to reach a nal
concentration of 0.5 mg/mL. The insoluble formazan was collected
and dissolved in DMSO and then measured using a microplate
reader (Thermo, Finland) at a wavelength of 490 nm. For the assay
of lactate dehydrogenase (LDH), the cells were incubated with
astin B for 24 h. The supernatant was collected, and LDH activity
was determined with a commercial kit (Jiancheng, Nanjing, China)
in accordance with the manufacturers instructions.
2.4. Measurement of intracellular ROS and GSH
The generation of intracellular ROS was assessed using the
ROS-specic uorescent dye 2,7-dichlorouorescein diacetate
(DCFH-DA; Beyotime, Haimen, China). L-02 cells were seeded at
an initial density of 1  105 cells/mL in 96-well plates. After 12 h
exposure to astin B, the cells were washed with PBS, loaded with
10 lmol/L DCFH-DA at 37 C for 30 min away from light, rinsed
three times with serum-free culture media, and measured at an

L. Wang et al. / Chemico-Biological Interactions 223 (2014) 19

excitation wavelength of 488 nm and an emission wavelength of


530 nm using a microplate uorescence reader (MD, spectramax
M3, USA). For GSH assay, the cells were cultured with astin B for
24 h and then harvested by centrifuging. The intracellular GSH
level was determined with commercially available kits (Jiancheng,
Nanjing, China) in accordance with the manufacturers
instructions.
2.5. Analysis of mitochondrial membrane potential (DWm)
The membrane potential assay is based on the JC-1 dye: JC-1
emits green uorescence when the DWm is relatively low. However, JC-1 aggregates and emits a red uorescence when the
DWm is high [16]. The assay was performed with a JC-1 kit in
accordance with the manufacturers instructions (Beyotime).
Briey, L-02 cells (1  105 cells/mL) in 6-well plates were separately treated with or without astin B for 24 h and were incubated
with JC-1 staining solution (5 lg/mL) for 20 min at 37 C in the
dark. The cells were rinsed twice with JC-1 staining buffer and
were then observed and photographed using an Olympus
uorescence microscope.
2.6. Measurement of cytochrome c (cyt c) content
After treatment with astin B for 24 h, the cells were lysed in
preparation buffer (250 mM sucrose, 20 mM HEPES-KOH (pH
7.4), 10 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA,
1 mM dithiothreitol, 2 lg/mL aprotinin and 1 mM phenylmethylsulfonyl uoride) for 10 min on ice followed by centrifugation at
10,000 rpm for 20 min at 4 C to separate the cytosolic and mitochondrial fractions [17]. Protein concentrations were measured
with a bicinchoninic acid protein assay kit (Beyotime). The contents of cyt c in the cytosol and mitochondria were determined
by ELISA kits (R&D Systems, Minnesota, USA) in accordance with
the manufacturers instructions.
2.7. Analysis of caspase activity
The activities of caspase-3 and caspase-9 were evaluated using
caspase-3 and caspase-9 activity assay kits (Beyotime). After a
24-h exposure, L-02 cells from 6-well plates were collected and
rinsed with cold PBS and later lysed using lysis buffer for 1 h on
ice. The cell lysates were centrifuged at 18,000 rpm for 10 min at
4 C. The assays were performed in 96-well plates by incubating
10 lL of the cell lysate supernatant in 80 lL reaction buffer
containing 10 lL Ac-DEVD-pNA (for caspase-3) and 10 lL
Ac-LEHD-pNA (for caspase-9). After further incubation at 37 C
for 4 h, the absorbance was measured using a microplate reader
(Thermo, Finland) at a wavelength of 405 nm.
2.8. Flow cytometric analysis of apoptotic cells
2.8.1. Sub-G1 peak
The apoptosis induced by astin B was determined using a cell
cycle and apoptosis analysis kit (Beyotime) in accordance with
the manufacturers instructions. In brief, approximately 1  105
L-02 cells were cultured in 6-well plates and separately treated
with or without astin B for 24 h. After treatment, the cells were collected and xed with 70% ethanol at 4 C overnight, centrifuged at
800 rpm for 5 min and stained with 500 lL of buffer, 25 lL of propidium iodide (PI), and 10 lL of RNase A in the dark for 30 min at
37 C. Next, the cells were analyzed using a ow cytometer (Becton
Dickinson, New Jersey, USA). The apoptotic cells with hypodiploid
DNA content were measured by quantifying the sub-G1 peak in the
cell cycle pattern. For each experiment, 10,000 events were
recorded per sample.

2.8.2. Annexin-V/PI staining


After 24 h treatment, the L-02 cells were gently trypsinized and
washed once with PBS, centrifuged at 800 rpm for 5 min, and
resuspended in 200 lL of binding buffer. After gentle pipetting,
the cells were stained by 3 lL of Annexin-V-FITC (AV) and 3 lL
of propidium iodide (PI) (BD Pharmingen, California, USA) for
15 min at room temperature in the dark and analyzed using a ow
cytometer (Becton Dickinson, New Jersey, USA). For each experiment, 10,000 events were recorded per sample.
2.9. Acridine orange (AO) staining of autophagic cells
Autophagic cells were detected by AO staining in accordance
with published procedures [18]. In brief, approximately 1  105
cells were seeded in 24-well plates and incubated with astin B
for 12 h, washed with PBS, and colored with AO (1 lg/mL)
(Generay, Shanghai, China) at 37 C for 1 min in the dark. The
cells were then observed and photographed by uorescence
microscopy.
2.10. Western blot analysis
The L-02 cells (approximately 6  105 in number) were
extracted with 100 lL of lysate buffer, which consists of 1 M
TrisHCl, 50% glycerine, 1.0% bromophenol blue, 10% SDS, and
b-mercaptoethanol. After boiling for 10 min, the extracted samples
were loaded at 20 lL/per lane, fractionated on 1215% trisglycine
precast gels, and then transferred to a PVDF membrane (Millipore,
Billerica, USA). The proteins were probed with primary antibodies
and HRP-labeled secondary antibodies and visualized using super
ECL detection reagent (Beyotime).
2.11. Animal experiments
2.11.1. Animals
Male ICR mice (68 weeks of age) were obtained from the
Laboratory Animal Center of Nanjing Qinglongshan (Nanjing,
China). They were maintained with free access to pellet food and
water in plastic cages at 21 2 C and kept on a 12-h light/dark
cycle. The care and treatment of these mice were in accordance
with the Provisions and General Recommendations of the Chinese
Experimental Animals Administration Legislation. This study was
approved by the Animal Ethics Committee of the School of Chinese
Materia Medica, China Pharmaceutical University.
2.11.2. Hepatotoxicity in mice
All of the mice were acclimatized for 3 days prior to experimental procedures. Two groups of mice (10 per group) were administered daily with astin B (10 mg/kg, suspended in distilled water)
or distilled water by gavage for 7 days. One hour after the last
administration, the mice were killed by cervical dislocation after
isourane inhalation, blood was collected from the orbital sinus
and centrifuged at 12,000 rpm for 5 min at 4 C to obtain serum
for the test of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities by commercial kits (Jiancheng, Nanjing,
China). Liver tissue was excised and xed in 10% phosphatebuffered formalin and embedded in parafn. Sections were
prepared and stained with hematoxylin and eosin (H&E) for histopathological analysis.
2.12. Statistical analysis
The data are expressed as the means SD (standard deviation)
from at least three independent experiments. Statistical analyses
were performed using one-way analysis of variance (ANOVA)

L. Wang et al. / Chemico-Biological Interactions 223 (2014) 19

followed by Students two-tailed t-test. Values of p < 0.05 were


considered to be statistically signicant.
3. Results
3.1. Astin B inhabits proliferation of L-02 cells
The potential cytotoxic effect of astin B was investigated by
incubating L-02 cells with astin B at different concentrations for
12, 24, and 48 h. The results of the MTT assay showed that astin
B decreased cell viability in a concentration-dependent and timedependent manner (Fig. 1B). After 48 h treatment, the cell viability
decreased to 39.22% at 60 lM. Leakage of LDH from cells is an indicator of cell necrosis. As shown in Fig. 1C, there was no signicant
leakage of LDH in cells after treatment with astin B at 15 and
30 lM for 24 h. Increased LDH leakage was observed only in cells
exposed to high concentrations of 45 and 60 lM.
3.2. Astin B induces oxidative stress

DCF fluorescence intensity

3.3. Astin B induces mitochondrial dysfunction


To assess whether astin B affects the functioning of mitochondria, we determined the changes in the mitochondrial membrane
potential (DWm). The results showed that astin B induced concentration-dependent DWm collapse in L-02 cells after incubation
with astin B for 24 h (Fig. 3A). Furthermore, the release of cyt c
from the mitochondria into the cytosol in the L-02 cells was also
detected. Exposure of cells to astin B for 24 h signicantly
increased the cyt c content of the cytosol fraction (Fig. 3B).
3.4. Astin B regulates Bcl-2/Bax expression and increases caspase-3
and -9 activity

To determine whether astin B induces oxidative stress, we


observed the effects of astin B on ROS production and GSH levels.
Compared with untreated cells, the intracellular ROS levels
increased signicantly when the cells were exposed to astin B for
12 h (Fig. 2A). In contrast, the GSH levels in the cells were reduced
in a concentration-dependent manner after treatment with astin B
for 24 h (Fig. 2B). Astin B promotes ROS production and decreases
GSH levels. Because JNK activity and oxidative stress are tightly
associated, we further observed the effects of astin B on JNK phosphorylation in cells treated with astin B at 30 lM for 0, 3, 6, 12, 24,
and 48 h. As shown in Fig. 2C, the phosphorylation of JNK was obviously enhanced after treatment with astin B for 3, 6, and 12 h.
Chlorogenic acid (CGA) is a well-known antioxidant and potential

5000

hepatoprotective [19]. After pretreatment with CGA for 12 h, the


cell viability of astin B-treated (15, 30, and 60 lM) groups
increased by 7.76% (p < 0.001), 9.24% (p < 0.01), and 6.03%
(p < 0.05), compared with the corresponding groups without pretreated by CGA (Fig. 2D). This may be evidenced from the reverse
side that astin B can cause oxidative stress and liver injury.

As apoptotic proteins, Bcl-2 is a powerful antagonist of apoptotic death programs, whereas Bax accelerates apoptotic death and
counters the death repressor activity of Bcl-2. The ratio of Bcl-2
to Bax determines the survival or death of the cells following an
apoptotic stimulus [20]. Treatment of L-02 cells with astin B significantly induced the expression of pro-apoptotic Bax and decreased
the expression of anti-apoptotic Bcl-2 in a concentrationdependent fashion (Fig. 4A). Thus, astin B signicantly increased
the Bax/Bcl-2 ratio (Fig. 4B) in a concentration-dependent manner,
leading to a state associated with apoptosis. Caspases are most
likely the most important effector molecules for the execution of
apoptosis [21]. When the L-02 cells were treated with astin B for

4000
3000
2000
1000
0

15

30

60

Astin B (M)
1.4

250
200

1.2

Relative viability

GSH (nmol/mg protein)

300

150
100

50

1.0
0.8
0.6
0.4
0.2
0.0

15

30

Astin B (M)

45

60

15
30
Astin B (M)

60

Fig. 2. Astin B induced oxidative stress in L-02 cells. (A) Cells were treated with astin B for 12 h, and intracellular ROS was assessed by the ROS-specic uorescent dye DCFHDA. (B) Cells were treated with astin B for 24 h, and the intracellular GSH level was determined using commercially available kits. (C) Cells were treated with astin B (30 lM)
at regular intervals, and phosphorylated JNK was determined using Western blot analysis. (D) Cells were pretreated with chlorogenic acid (CGA+, 100 lM) for 12 h, following
treated with astin B for 24 h, and cell viability was determined by the MTT assay. Data in (A, B, and D) are expressed as the mean SD from three independent experiments
with n = 46. p < 0.05 compared with the control group (0 lM), #p < 0.05 compared with the corresponding CGA group. The results shown in C are representative of three
independent experiments.

L. Wang et al. / Chemico-Biological Interactions 223 (2014) 19

B
400

Cyt c (ng/mg protein)

350
300

Mitochondria

Cytosol

250

* *

200

* *

150

* *

100

50
0

15

30

45 60

15

30 45

60

Astin B (M)
Fig. 3. Astin B decreased the mitochondrial membrane potential (DWm) and promoted cytochrome c release. (A) L-02 cells were treated with astin B for 24 h and
subsequently incubated with JC-1 dye for 20 min, then observed and photographed by an Olympus uorescence microscope. The proportion of green uorescence emission
represents the DWm collapse degree. (B) Cells were treated with astin B for 24 h, and the content of cytochrome c was measured with ELISA kits. The results shown in A are
representative of three independent experiments. Data in (B) are expressed as the mean SD from three independent experiments with n = 4. p < 0.05 compared with the
control group (0 lM). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

Ratio of Bax/Bcl-2

0
0

15

30

45

60

Astin B (M)
4

Caspase-9

Fold of control

Fold of control

*
*

Caspase-3

3
2
1
0

0
0

15

30

45

60

15

1.4

zVAD -

1.2

Relative viability

30

45

60

Astin B (M)

Astin B (M)

zVAD +

0.8
0.6
0.4
0.2
0
0

15

30

60

Astin B (M)
Fig. 4. Astin B regulated Bcl-1/Bax expression and increased activities of caspases-9 and -3 in L-02 cells. Cells were treated with astin B for 24 h: (A) Bax and Bcl-2 expressions
were determined by Western blot, and (B) the ratio of Bax/Bcl-2 was calculated by densitometry. Enzymatic activities of (C) caspase-9 and (D) caspase-3 were measured using
commercial kits. (E) Cell viability in the presence of z-VAD-fmk (z-VAD+, 50 lM), a pan-caspase inhibitor, was determined by the MTT assay. The results shown in (A) are
representative of three independent experiments. The data in B-E are expressed as the mean SD from three independent experiments with n = 3. p < 0.05 compared with
the control group (0 lM) in (BD) or with the corresponding z-VAD-group in E.

L. Wang et al. / Chemico-Biological Interactions 223 (2014) 19

24 h, the activities of caspases-3 and -9 were increased in concentration-dependent manners (Fig. 4C and D). Co-treatment of astin B
with z-VAD-fmk (a pan-caspase inhibitor) could increase the viability of the L-02 cells to some extent, compared with the groups
that were only treated by astin B with corresponding concentrations (Fig. 4E). These results suggest that astin B most likely
mediates apoptotic cell death and that they do so mainly in a
caspase-dependent manner.
3.5. Astin B induces apoptotic cell death
To conrm whether astin B induced cell death through apoptosis, we analyzed the sub-G1 peaks, i.e., the population of apoptotic
cells, of the L-02 cells. After treatment of the cells with astin B at 0,
15, 30, 45, and 60 lM for 24 h, the percentage of the sub-G1 peak
gradually increased in a concentration-dependent manner from
4.09% to 14.22% (Fig. 5A and B), indicating apoptotic cell death
induced by astin B in L-02 cells. This nding was further evidenced
by the Annexin-V/PI staining method, which can clearly discriminate among the four types of cells, i.e., unaffected cells (AV/
PI), early apoptotic cells (AV+/PI), early necrotic and late apoptotic cells (AV+/PI+), and late necrotic cells (died without apoptosis) (AV/PI+). After treatment with astin B at same
concentrations for 24 h, the early apoptotic L-02 cells increased
obviously in a concentration-dependent fashion from 8.03% to
47.12%, along with lower increases of necrotic and late apoptotic
cells from 4.64% to 16.33% (Fig. 5C and D). These results indicate
that apoptosis is one of the major ways for astin B to induce L-02

cell death, which may be one of the main hepatotoxic components


in RAT.
3.6. Astin B induces autophagy against apoptosis
Autophagy is important for cell death decisions and can protect
cells by preventing them from undergoing apoptosis [22]. Therefore, a method of AO staining was employed to detect autophagic
effects in L-02 cells. As shown in Fig. 6A, after exposure to astin
B for 12 h, autophagic vacuoles were visible in the cells. Furthermore, the expressions of LC3-I/LC3-II and p62, both key indicators
for autophagosome formation [23,24], were examined by Western
blot analysis. The results showed that the treatment of L-02 cells
with astin B signicantly enhanced LC3-II and reduced p62 expression (Fig. 6B). These data indicate that the stimulus of astin B
induces both autophagy and apoptosis in hepatocytes. In addition,
the connection between autophagy and apoptosis was investigated
with astin B-induced L-02 cells by co-treatment with 3-MA (an
autophagy inhibitor) or z-VAD-fmk (a caspase inhibitor).
Compared with treatment with astin B alone, co-treatment with
3-MA obviously attenuated LC3-II expression, rapidly enhanced
caspase-3 expression, and signicantly reduced cell viability in
L-02 cells (Fig. 6C and D). On the other hand, co-treatment with
z-VAD-fmk resulted in an accrual of autophagic pathway evidenced by increased LC3-II and decreased p62 (Fig. 6E), which
may partially explain why the cell viability was only increased
slightly on blocking apoptosis (Fig. 4E). These results indicated that
astin B-induced LC3-II enhancement and p62 redaction were the

12

10

60

Cell number (%)

Cells of sub-G1 peak (%)

16

14

8
6

40

10
0

30

Astin B (M)

45

60

AV+PI-

20

0
15

*
*

AV+PI+

30

2
0

50

*
0

15

**
30

*
45

60

Astin B (M)

Fig. 5. Astin B increased the apoptotic L-02 cells. Cells were treated with astin B for 24 h, and apoptotic cells were determined by ow cytometry. (A and B) The population of
sub-G1 peaks (indicated by an arrow) was measured by propidium iodide staining, and (C and D) apoptotic and necrotic cells were claried by Annexin V-FITC (AV) and
propidium iodide (PI) staining. The results shown in (A) and (C) are representative of three independent experiments. The data in (B) and (D) are expressed as the mean SD
from three independent experiments. p < 0.05 compared with the control group (0 lM).

L. Wang et al. / Chemico-Biological Interactions 223 (2014) 19

1.2

Relative viability

D
P 0.05

0.8
0.6
0.4
0.2
0

E
B

Fig. 6. Astin B induced autophagy in L-02 cells to protect from apoptosis. (A) After treatment with astin B (30 lM) for 12 h, autophagy in the L-02 cells was detected by
acridine orange (AO) staining, and orange-colored vacuoles indicated positive results, Earles balanced salt solution (EBSS) as positive control. (B) After treatment with astin B
(15, 30, 60 lM) for 12 h, the levels of p62 and LC3-I/LC3-II in L-02 cells were determined by Western blot analysis. (C and D) L-02 cells were exposed to astin B (60 lM) with or
without the presence of the autophagy inhibitor 3-MA (2 mM) for 12 h, the levels of LC3-I/LC3-II and procaspase-3/cleaved caspase-3 were determined by Western blot
analysis, and cell viability was measured by the MTT assay. (E) L-02 cells were treated with astin B (60 lM) with or without the presence of the pan-caspase inhibitor z-VADfmk (50 lM) for 12 h, the levels of p62 and LC3-I/LC3-II were determined by Western blot analysis. The results shown in A are representative of three independent
experiments. The results shown in (B, C, and E) are representative of three independent experiments. The data in (D) are expressed as the mean SD from three independent
experiments.

B
160

Enzyme activity (U/L)

140

ALT

**

120

AST
100

**

80
60
40
20
0

Control

Astin B

Fig. 7. Hepatotoxicity of astin B in mice. Ten mice were orally administered with vehicle (control group) or astin B at 10 mg/kg once daily for 7 consecutive days. (A) ALT and
AST levels in serum were determined by commercial kits. (B) Slices of liver were stained with H&E for histopathological analysis. The data in (A) are expressed as the
mean SD, p < 0.01 compared with the control group; the results shown in (B) are representative of ve independent experiments at a magnication of 200.

L. Wang et al. / Chemico-Biological Interactions 223 (2014) 19

real events associated with autophagosomes and that astin Binduced apoptotic cell death will be accelerated in the absence of
autophagy in hepatocytes.

3.7. Astin B induces liver injury in mice


To investigate whether astin B induces liver injury in vivo, we
observed the effects of orally administered astin B on the liver cells
in mice. Compared to the control group, the serum levels of ALT
and AST in mice treated with astin B were elevated about 11.9
and 2.26 times, respectively (Fig. 7A). The liver injury of astin B
was further conrmed by histopathological examination, as
evidenced by diffuse hydropic degeneration of hepatocytes with
moderate inammatory cell inltration (Fig. 7B). The results
indicated that, consistent with its action in vitro, astin B could also
induce liver injury in vivo.

4. Discussion
Our previous work suggested that pentapeptides, especially the
cyclic ones, were most likely the principal toxic components in RAT
[14]. Astins are a class of halogenated cyclic pentapeptides contained in RAT. Although astins have been reported to kill tumor
cells and prevent colitis through regulation of apoptosis [1113],
our results clearly showed that astin B, one of the cyclic pentapeptides, induced hepatic cell death mainly by ROS-associated apoptosis via a mitochondria-dependent pathway. Such cell death will
lead to liver injury, which was evidenced by the in vivo experiment.
On the other hand, astin B also positively regulated autophagy, and
this action partially attenuated apoptotic cell death.
MTT assays showed that astin B effectively inhibited cell proliferation and survival. The inhibitory percentage reached 60.78% at
60 lM after 48 h exposure. Apoptosis and necrosis are the most
frequent events in xenobiotic metabolism-induced liver injury
[25]. Astin B substantially increased cell death, while the leakage
of LDH, an indicator of necrosis, only moderately increased in cells
that were exposed to high concentrations. This nding indicates
that apoptosis is most likely the major event that is responsible
for the cell death.
Uncontrolled ROS production and/or decreased antioxidant
defenses result in oxidative stress, which has been considered to
be an important pathogenic element for the initiation of hepatotoxicity [4,6]. GSH is a major antioxidant that guards cells against
oxidative injury by diminishing ROS. Therefore alterations in the
GSH level can be monitored as an indication of oxidative stress
in cells [26]. Astin B treatment enhanced ROS production with
downregulation of GSH levels in L-02 cells, indicating that oxidative stress occurred following the astin B stimulus. Changes in
ROS and GSH act as intracellular signals and can evoke inammation by the regulation of transcription factor molecules through
JNK activation [27]. Consistent with this view, we observed
enhanced pJNK expression when the cells were exposed to astin
B. Moreover, the inhibited cell proliferation and survival by astin
B could be reversed by antioxidants, such as CGA, to some extent.
All these alterations indicate that the astin B stimulus induces
ROS-associated inammation in hepatocytes.
Given that the mitochondria are the major intracellular source
of ROS, we wondered whether ROS-associated inammation would
impair mitochondrial function. Our experiments showed that astin
B induces the collapse of DWm with an increased release of cyt c
from the mitochondria into the cytosol. This result suggests that
mitochondrial dysfunction results in the opening of the mitochondrial permeability transition pores, leading to the release of cyt c
from the mitochondria.

In view of the link between mitochondrial dysfunction and


apoptosis, the participation of the Bcl-2 family (which contains
both pro- and anti-apoptotic proteins) is a key event because of
the regulative effects of these proteins on mitochondrial dysfunction during apoptosis. In such regulation, Bcl-2 is an anti-apoptotic
member, while Bax is a pro-apoptotic multidomain effector protein
[28]. Increased pro-apoptotic proteins can actively permeabilize
the outer mitochondrial membrane and promote the release of
intermembrane space proteins, such as cyt c, into the cytosol. Cyt
c is required for the activation of caspase-9. Generally, activated
caspase-9 is involved in the mitochondrial pathway, and it activates downstream caspase-3, thereby triggering cell apoptosis
[29]. The increased ratio of Bax/Bcl-2 observed in cells treated with
astin B indicates that astin B induces mitochondrial outer membrane permeabilization, which discharges cyt c into the cytosol.
Furthermore, the increased activities of caspases-9 and -3 observed
in treated cells suggest that the astin B-induced apoptosis may follow a caspase-dependent intrinsic mitochondrial pathway. The initiation of apoptosis induced by astin B was further characterized
by an increased sub-G1 peak population during the cell cycle and
a predominant population of early apoptotic cells among the dead
cells.
Autophagy functions as a cytoplasmic quality control mechanism to remove protein aggregates and damaged organelles.
Although autophagy has been observed in many dying cells, it is
generally accepted that autophagy is a pro-survival and protective
pathway [30,31]. Apoptosis and autophagy are genetically regulated processes that regulate cell fate. The functional relationship
between apoptosis and autophagy is quite complex. In several scenarios, autophagy prevents cell death and de facto suppresses
apoptosis; and it also appears that similar stimuli can induce either
apoptosis or autophagy [32]. AO-stained autophagic vacuoles,
enhanced LC3-II and reduced p62 in L-02 cells treated with astin
B conrmed that astin B can also induce autophagy in hepatocytes.
After co-treatment with the autophagy inhibitor 3-MA, decreased
cell viability and dramatically increased expression of cleaved
caspase-3 were observed. Conversely, co-treatment with caspase
inhibitor z-VAD-fmk led to an enhanced autophagy for astin
B-treated cells evidenced by increased LC3-II and decreased p62
expression. These results indicate that apoptosis and autophagy
induced by astin B were interacted and that autophagy could
effectively protect the cells from B-induced cell apoptosis. The
protective pathway of autophagy, i.e., whether autophagy is a secondary result of cell death that functions to remove damaged
organelles or if it is directly induced by astin B in an apoptosisindependent manner, remains to be determined.
In summary, our studies suggest that astin B will provoke oxidative stress-associated inammation and induce hepatocyte
apoptosis through a mitochondria-dependent pathway, leading to
liver injury. However, astin B will also induce autophagy to protect
cells from apoptosis and to alleviate hepatic injury to some extent.
Furthermore, we found that an astin-rich fraction (astins were
71.2% of the relative content) [14] induces apoptosis in the same
fashion (data not shown). As a result, astins (including astin B)
can be considered to be the major hepatotoxic substances in the
herbal medicine derived from A. tataricus. Owing to their special
and interesting structures, previous studies of astins focused on
their biological activities, and their toxic or side effects were often
overlooked. We hope that our nding will be helpful for providing
advice regarding drug safety for the clinical use of RAT and the
development of new pharmaceuticals.

Conict of Interest
The authors declare that there are no conicts of interest.

L. Wang et al. / Chemico-Biological Interactions 223 (2014) 19

Transparency Document
The Transparency document associated with this article can be
found in the online version.

Acknowledgments
This work was nancially supported by the National Natural
Science Foundation of China (30772702) and the National New
Drug Innovation Major Project of China (2011ZX09307-002-02).
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