Astin B, a cyclic pentapeptide from Aster tataricus, induces apoptosis and autophagy in human hepatic L-02 cells

Chemico-Biological Interactions 223 (2014) 1–9

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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

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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 identification 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 inflammation 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 fluorescence, 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. Ó 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 classified into several modes such as apoptosis, necrosis, necroptosis, autophagy, and cornification [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: [email protected] (C.-F. Zhang), [email protected] (M. Zhang). http://dx.doi.org/10.1016/j.cbi.2014.09.003 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

inflammation, 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 A–I, 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 [11–13]. However, our previous studies discovered the potent hepatotoxicity of RAT in mice, and a

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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 identification 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.). Earle’s balanced salts solution (EBSS), 3-methyladenine (3-MA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma–Aldrich (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 humidified atmosphere (5% CO2). The medium was renewed every 2 days until the cells were grown to confluence. 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 final 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 manufacturer’s instructions. 2.4. Measurement of intracellular ROS and GSH The generation of intracellular ROS was assessed using the ROS-specific fluorescent dye 2,7-dichlorofluorescein 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) 1–9

excitation wavelength of 488 nm and an emission wavelength of 530 nm using a microplate fluorescence 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 manufacturer’s instructions. 2.5. Analysis of mitochondrial membrane potential (DWm) The membrane potential assay is based on the JC-1 dye: JC-1 emits green fluorescence when the DWm is relatively low. However, JC-1 aggregates and emits a red fluorescence when the DWm is high [16]. The assay was performed with a JC-1 kit in accordance with the manufacturer’s instructions (Beyotime). Briefly, 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 fluorescence 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 fluoride) 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 manufacturer’s 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 manufacturer’s 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 fixed 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 flow 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.

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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 flow 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 fluorescence 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 Tris–HCl, 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 12–15% tris–glycine 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 (6–8 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 isoflurane 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 fixed in 10% phosphatebuffered formalin and embedded in paraffin. 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)

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followed by Student’s two-tailed t-test. Values of p < 0.05 were considered to be statistically significant. 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 significant 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 significantly 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 significantly 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

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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.

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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 significantly 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

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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-specific fluorescent 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 = 4–6. ⁄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.

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Astin B (µM) 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 fluorescence microscope. The proportion of green fluorescence 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 figure legend, the reader is referred to the web version of this article.)

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Astin B (µM) 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 (B–D) or with the corresponding z-VAD-group in E.

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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 confirm 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 finding 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 significantly 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 significantly 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

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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 flow 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 clarified 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).

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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, Earle’s 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.

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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 five independent experiments at a magnification of 200.

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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 confirmed by histopathological examination, as evidenced by diffuse hydropic degeneration of hepatocytes with moderate inflammatory cell infiltration (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 [11–13], 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 finding 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 inflammation 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 inflammation in hepatocytes. Given that the mitochondria are the major intracellular source of ROS, we wondered whether ROS-associated inflammation 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 confirmed 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 inflammation 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 finding will be helpful for providing advice regarding drug safety for the clinical use of RAT and the development of new pharmaceuticals.

Conflict of Interest The authors declare that there are no conflicts of interest.

L. Wang et al. / Chemico-Biological Interactions 223 (2014) 1–9

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

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