Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
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.
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
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).
5000
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
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.
B
400
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.
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
12
10
60
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).
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
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.
real events associated with autophagosomes and that astin Binduced apoptotic cell death will be accelerated in the absence of
autophagy in hepatocytes.
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.
Conict of Interest
The authors declare that there are no conicts of interest.
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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