Esculetin induces apoptosis in human gastric cancer cells through a cyclophilin D-mediated mitochondrial permeability transition pore associated with ROS

Esculetin induces apoptosis in human gastric cancer cells through a cyclophilin D-mediated mitochondrial permeability transition pore associated with ROS

Chemico-Biological Interactions 242 (2015) 51e60 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.els...

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Chemico-Biological Interactions 242 (2015) 51e60

Contents lists available at ScienceDirect

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

Esculetin induces apoptosis in human gastric cancer cells through a cyclophilin D-mediated mitochondrial permeability transition pore associated with ROS Hui Pan a, *, 1, Bao-Hui Wang b, 1, Wang Lv a, Yan Jiang b, Lei He a a b

The First Affiliated Hospital, College of Medicine, Zhejiang University, Qingchun Road 79, Hangzhou, China Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 May 2015 Received in revised form 20 August 2015 Accepted 16 September 2015 Available online xxx

Esculetin is a coumarin derivative from natural plants that has been commonly used as a folk medicine and has been reported to have beneficial pharmacological and biochemical activities; however, the mechanism by which esculetin prevents human gastric cancer cell growth is still largely unknown. In this study, we investigated the effect of esculetin on human gastric cancer cells and explored the cell death mechanism. Our data indicated that esculetin inhibited the growth of human gastric cancer cells in a dose- and time-dependent manner and apoptosis was the main cause of decreased cell viability in esculetin-treated cells. Additionally, esculetin treatment increased the activity of caspase-9 and caspase3, and resulted in the appearance of the PARP cleavage product; and esculetin-induced cell death and apoptosis was decreased by pretreatment with CsA and NAC, but not BA; these results demonstrate that esculetin induced apoptosis via the caspase-dependent mitochondrial pathway in human gastric cancer cells in which cyclophilin D mediated the cytotoxic action by triggering the opening of the mitochondrial permeability transition pore; and the generation of ROS not only was a consequence of mitochondrial dysfunction, but also triggered esculetin-induced apoptosis. These results reveal a novel mechanism of esculetin on gastric cancer cells and suggest that esculetin could be a novel agent in the treatment of gastric cancer. © 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Esculetin Apoptosis Cyclophilin D ROS Gastric cancer cells

1. Introduction Gastric cancer is the fourth most common malignancy in the world in numbers of cases and the second leading cause of cancer deaths in both sexes worldwide [1,2]. The highest mortality rates are estimated in Eastern Asia (28.1 per 100,000 men, 13.0 per 100,000 women), and the lowest in Northern America (2.8 and 1.5, respectively). High mortality rates are also present in both sexes in Central and Eastern Europe and Central and South America [1]. Of the patients that present with earlier stages of the disease, more than 50% undergo surgery, but even after a curative resection, 60% of these patients eventually relapse and die due to their disease [3,4], which highlights the urgent need for novel, effective therapeutic approaches.

* Corresponding author. E-mail address: [email protected] (H. Pan). 1 Hui Pan and Bao-Hui Wang contributed equally. http://dx.doi.org/10.1016/j.cbi.2015.09.015 0009-2797/© 2015 Elsevier Ireland Ltd. All rights reserved.

Several Chinese herbs and their active components have been reported to effectively inhibit gastric cancer cell proliferation [5,6]. Coumarins comprise a group of aromatic lactones of phenolic compounds and are composed of fused benzene and a-pyrone rings [7]. Coumarins are widely distributed in the plant kingdom; more than 1300 coumarins have been identified in natural sources, especially in green plants [8,9]. This class of compounds influences the formation and scavenging of reactive oxygen species and takes part in processes involving free radical-mediated injuries [10,11]. Coumarins vary widely in structure due to the various types of substitutions in their basic rings, which can influence their biological activity [12,13]. Coumarins are reported to exhibit a wide range of pharmacological properties, including anti-inflammatory, antioxidant, antimutagenicity, and antibacterial effects; stimulate immunity; and inhibit tumor growth and metastasis [14e16]. Esculetin is a coumarin derivative that is found in various natural plants, such as Artemesia scoparia (Redstem Wormwood), Artemesia capillaris (Capillary Wormwood), and Ceratostigma willmottianum (Chinese Plumbago), and in the leaves of Citrus limonia

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(Chinese lemon) [17,18], which are commonly used as folk medicines and have been reported to have beneficial pharmacological and biochemical activities. Esculetin has been shown to inhibit the lipoxygenase and cyclooxygenase pathways of arachidonate metabolism [19]. Among other important pharmacological activities, esculetin protects hamster lung fibroblasts from lipid peroxidation [16] and DNA against oxidative stress [19]; promotes analgesic [20] and immunomodulatory processes [21]; inhibits the synthesis of leukotriene B4 and thromboxane B2 [11], platelet aggregation [22], and matrix metalloproteinases production [23]; and limits the growth of several human cancer cell lines [24,25]; however, there is little information available concerning the ability of esculetin to inhibit gastric cancer. Although the induction of cytotoxicity by esculetin has been observed in some cancer cell lines, the mechanisms by which esculetin induces cytotoxicity are generally unknown. In the present study, we investigated the effects of esculetin on human gastric cancer cells and further examined the cell death mechanism. Our observations demonstrated that esculetin-induced cytotoxicity was attributed to apoptosis in human gastric cancer cells and that this activity was closely associated with the cyclophilin D (CypD)-mediated mitochondrial permeability transition pore (MPTP). These preclinical studies suggest that esculetin could be a potential medicine for the treatment of gastric cancer.

2.4. Apoptosis assays The apoptotic rates were analyzed by flow cytometry using an annexin V-FITC apoptosis detection kit (SigmaeAldrich) according to the manufacturer's instructions. Briefly, the cells were seeded in six-well plates (5  105 cells/well) and incubated with esculetin for 24 h, and then, the cells were harvested. The cells were washed twice with ice-cold PBS and evaluated for apoptosis by double staining with a fluorescein isothiocyanate (FITC)-conjugated antiannexin V antibody and propidium iodide in binding buffer using a flow cytometer (Becton Dickinson). To detect DNA strand breaks, a TUNEL assay was performed using an in situ DNA fragmentation assay kit (Biovision) according to the manufacturer's instructions. Briefly, the cells were seeded in six-well plates (5  105 cells/well) and incubated with esculetin for 24 h, and then the collected cells were fixed in 4% paraformaldehyde. After three washings, the cells were added to 70% ice-cold ethanol followed by an incubation with a mixture of BrdUTP (bromolated deoxyuridine triphosphate nucleotides) and TdT enzymes for 1 h at 37  C. The cells were incubated with the anti-BrdU-Red antibody in the dark for 30 min at room temperature. The stained cells were analyzed using flow cytometry (Becton Dickinson). 2.5. Caspase assays

2. Material and methods 2.1. Material and reagents Esculetin (with >98% purity), 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), z-VAD-fmk (z-VAD), Nacetyl-l-cysteine (NAC) and dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma. Cyclosporin A (CsA), bongkrekic acid (BA), and antibodies specific for cyclophilin D (CypD) and adenine nucleotide translocator (ANT) were purchased from Santa Cruz Biotechnology. Antibodies specific for Bcl-2, Bcl-xL, Bax, Bak, Bad, XIAP, cleaved PARP, cytochrome c and b-actin were purchased from Cell Signaling Technology. All of the other chemicals were of the highest purity available.

2.2. Cell lines and cell culture The human normal gastric epithelial cell line GES-1, the human gastric cancer cell lines SGC-7901, MGC-803, BGC-823 were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All of the cell lines were routinely cultured in RPMI 1640 medium (Gibco), which contained 10% fetal calf serum (Gibco), 100 units/ml penicillin, and 100 units/ ml streptomycin, in a humidified cell incubator at 37  C with an atmosphere of 5% CO2.

2.3. Cell viability assay Cell viability was measured by the MTT method. Briefly, exponentially growing cells were seeded in 96-well plates and cultured until 70% confluence and were then incubated with esculetin at various concentrations for the indicated times. MTT was then added to each well and the cells were incubated for an additional 4 h at 37  C. The formazan precipitate was dissolved in 150 ml of DMSO, and the absorbance at 490 nm was measured using a microplate reader (Thermo). Each test was repeated at least three times.

Caspase-3, -8, and -9 activities were measured using a caspase activity kit assay (Santa Cruz Biotechnology) according to the manufacturer's instructions. Briefly, after the cells were treated with esculetin for 24 h, the cell lysate from 1  106 cells was incubated at 37  C for 2 h with 200 mM DEVD-pNA (caspase-3 substrate), IETD-pNA (caspase-8 substrate) or LEHD-pNA (caspase9 substrate). The samples were read at 405 nm in a microplate reader (Thermo) and expressed as a fold increase from the basal level. In the caspase inhibitor assay, the cells were treated with esculetin at the indicated concentrations for 24 h, with or without a 100 mM z-VAD-fmk pretreatment for 2 h, and then apoptosis was determined using the TUNEL assay as described above. 2.6. Determination of cellular reactive oxygen species Reactive oxygen species (ROS) were determined using a flow cytometer and DCFH-DA staining. The cells were incubated with 10 mM DCFH-DA at 37  C for 30 min. After incubation with the fluorochrome, the cells were washed with phosphate buffered saline and immediately analyzed by flow cytometry. 2.7. Detection of the mitochondrial membrane potential The mitochondrial membrane potential (MMP) was detected using a JC-1 assay kit (Beyotime, China) according to the manufacturer's instructions. The cells were cultured on coverslips in a 24-well plate. After the cells were treated with esculetin for 24 h, the cells were incubated with JC-1 for 20 min and observed using an Olympus FV1000 confocal microscope. In the control group, the JC-1 dye concentrated in the mitochondrial matrix where it formed red fluorescent aggregates. The green fluorescence represents the monomeric form of JC-1, which appeared in the cytosol after mitochondrial membrane depolarization. The level of mitochondrial depolarization was expressed as the red/green fluorescence intensity ratio. 2.8. Western blot analysis Western blot analysis was performed using standard methods.

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Briefly, approximately 30 mg of lysed proteins were separated by 10e15% sodium dodecyl sulfate (SDS) PAGE, transferred to a nitrocellulose blotting membrane (Bio-Rad) and blocked for 1 h in blocking buffer (5% bovine serum albumin solution and 0.1% Tween 20 in tris-buffered saline (TBST)). After three washes in TBST, the membrane was incubated with the primary antibody overnight at 4  C. After three washing steps in TBST, the blots were subjected to the appropriate secondary antibodies conjugated with Alexa [email protected] 680 (Jackson Immuno Research Laboratories, USA) for 1 h in blocking buffer. After three washes in TBST for 15 min, the proteins were visualized by an Odyssey® Imager (LI-COR, USA). 2.9. Transient transfection with small interfering siRNA siRNA targeting CypD were purchased from Santa Cruz Biotechnology. The cells were transfected with the corresponding siRNA of the target gene or ascrambled control siRNA using lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. The Knockdown efficiency was assessed by western blot after 48 h of transfection. 2.10. Statistical analysis All of the data were expressed as the mean ± standard deviation (SD) from at least three independent experiments and analyzed by one-way ANOVA using SPSS (version 16.0). A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Esculetin inhibits the growth of human gastric cancer cells through the apoptosis pathway To identify the therapeutic potential of esculetin, MGC-803, SGC-7901, and BGC-823 cells were cultured with the indicated concentrations of esculetin for 24, 48, and 72 h, and then cell viability was determined using an MTT assay. Esculetin inhibited

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the growth of cells in a dose- and time-dependent manner (Fig. 1CeE). The 50% inhibitory concentrations (IC50) for the MGC803 cells were 40.2, 35.1 and 30.8 mM after treatment with esculetin for 24, 48, and 72 h, respectively; the IC50 in the SGC-7901 cells and BGC-823 cells after treatment with esculetin were 57.1 and 76.2 mM (for 24 h), 48.1 and 63.4 mM (for 48 h), 38.6 and 50.3 mM (for 72 h), respectively. In contrast, only a small percentage of cell death was found in GES-1 cells after treatment with esculetin (Fig. 1B). These results suggest that human gastric cancer cells are more susceptible to esculetin-induced cell death compared with normal GES-1 cells. To determine whether the cytotoxicity of esculetin was caused by apoptosis, annexin V-FITC/PI double staining and TUNEL assay were performed. As shown in Fig. 2A, compared to the control, the MGC-803 cells treated with 50 mM esculetin had an increased percentage of early apoptotic cells (Annexin V positive but PI negative cells) from 3.85% to 42.85%, and an increased percentage of late apoptotic/necrotic cells (Annexin V and PI double-positive cells) from 3.36% to 32.40%. In the TUNEL assay, flow cytometric analysis showed that esculetin treatment of human gastric cancer cells caused a dose- and time-dependent increase in the percentage of apoptotic cells (TUNEL-positive cells) (Fig. 2BeD). These results demonstrate that apoptosis was the main cause of decreased cell viability in esculetin-treated cells.

3.2. Esculetin induces caspase-dependent apoptosis in human gastric cancer cells To determine whether esculetin-induced apoptosis was dependent on caspase activation, human gastric cancer cells were cultured in the presence and absence of the caspase inhibitor, zVAD-fmk. As shown in Fig. 3A, z-VAD effectively attenuated esculetin-induced cytotoxicity. In addition, the TUNEL assay showed that esculetin-induced apoptosis was largely inhibited upon z-VAD treatment in human gastric cancer cells (Fig. 3B). We further demonstrated that caspase-3, the major effect indicator in the apoptotic pathway [26], was involved in esculetin inducedcytotoxicity. The data revealed that esculetin treatment resulted

Fig. 1. Time- and dose-dependent effects of esculetin on cell viability. (A) Chemical structure of esculetin. (B) Effect of esculetin on the cell growth inhibition of normal GES1 cells. (CeE) Esculetin inhibits the growth of MGC-803(C), SGC-7901(D) and BGC-823(E) cells in a dose- and time-dependent manner. Cell viability was detected by the MTT assay. E 12.5, esculetin 12.5 mM; E 25, esculetin 25 mM; E 50, esculetin 50 mM; E 100, esculetin 100 mM; E 150, esculetin 150 mM. Data represents the mean ± SD of three independent experiments (*p < 0.05 versus control).

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Fig. 2. Esculetin induces apoptosis in human gastric cancer cells. (A) Images of flow cytometry show that there was clear apoptosis detected in MGC-803 cells after treatment with esculetin for 24 h, and the level of apoptosis was determined by annexin V-FITC/PI double staining assay. (BeD) Bar graphs show that the cell apoptotic rates increase significantly in human gastric cancer cells after treatment with esculetin for 24 h in a dose-dependent manner and with 50 mM esculetin in a time-dependent manner. The cell apoptotic rates were detected by TUNEL assay. E 12.5, esculetin 12.5 mM; E 25, esculetin 25 mM; E 50, esculetin 50 mM. Data represents the mean ± SD of three independent experiments (*p < 0.05 versus control).

in a marked increase in caspase-3 activity after 24 h in a dosedependent manner (Fig. 3C). Correspondingly, the amount of cleaved PARP, as demonstrated by the appearance of the 85-kDa cleavage product, increased after esculetin treatment of human gastric cancer cells in a dose-dependent manner (Fig. 3D). Altogether, these data implied that esculetin induces cytotoxicity in human gastric cancer cells through an apoptotic mechanism dependent on caspase activation. 3.3. Esculetin-induced apoptosis is mediated through the mitochondrial pathway in MGC-803 cells Caspases play critical roles in the process of cell apoptosis. Caspase-8 and casepase-9 are the apical proteases in the receptor

mediated pathway and the mitochondrial mediated pathway, respectively [27,28]. We examined the activity of caspase-3, -8 and -9. As shown in Fig. 4A, although there was little increase in the activity of caspase-8, esculetin treatment increased the activity of caspase-9 and caspase-3 in a dose-dependent manner. Moreover, western blot analysis revealed that the relative amount of cytochrome c in the cytosol of the cells treated with esculetin dramatically increased in a dose-dependent manner compared to vehicle-treated cells (Fig. 4B). To confirm whether pro-apoptotic and anti-apoptotic regulatory proteins were involved in esculetininduced apoptosis in MGC-803 cells, we analyzed the expression of Bcl-2 family members and inhibitors of apoptotic proteins by western blot analysis. As shown in Fig. 4C, esculetin dosedependently enhanced the expression of Bax and Bak (pro-

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Fig. 3. Esculetin induces caspase-dependent apoptosis in human gastric cancer cells. (A) The bar graphs that show pretreatment with 0.1 mM caspase inhibitor, z-VAD, for 2 h reversed esculetin-induced cell death in MGC-803, SGC-7901 and BGC-823 cells. The cells were incubated with esculetin for 24 h, and cell viability was detected by MTT assay. (B) The bar graphs show that z-VAD decreased esculetin-induced apoptosis rates. The cell apoptotic rates were detected by the TUNEL assay. (C) The bar graphs show that the activity of caspase-3 increased significantly after treatment with esculetin. (D) Images of western blot showing the expression of cleaved PARP after treatment with esculetin for 24 h (Data represent similar results from three independent experiments). E 12.5, esculetin 12.5 mM; E 25, esculetin 25 mM; E 50, esculetin 50 mM. The data represents the mean ± SD of three independent experiments (*p < 0.05 versus control).

apoptotic regulatory proteins) and down-regulated the expression of Bcl-2 and Bcl-xL (anti-apoptotic regulatory proteins). We also investigated the effect of esculetin on the mitochondrial

integrity. As shown in Fig. 5A, esculetin dose-dependently enhanced the expression of CypD, while did not affect ANT expression. Moreover, esculetin-treated MGC-803 cells showed a

Fig. 4. Esculetin-induced apoptosis is mediated through the mitochondrial pathway in MGC-803 cells. (A) The bar graphs show the increase of caspase activation in esculetininduced apoptosis in MGC-803 cells. After treatment with esculetin for 24 h, the cytosolic fraction of the cells was analyzed for changes in the activity of caspase-3, -8, and -9 (Data represents the mean ± SD of three independent experiments, *p < 0.05 versus control). (B) Determination of cytochrome c release in MGC-803 cells. After treatment with esculetin for 24 h, the cytosolic fraction was isolated and the content of cytochrome c was examined by western blot analysis. (C) The images of western blot show the effect of esculetin on apoptotic proteins. After treatment with esculetin for 24 h, whole-cell lysates were subjected to western blot analysis to assess the expression of Bcl-2 family proteins and XIAP. Data represent similar results from three independent experiments. E 12.5, esculetin 12.5 mM; E 25, esculetin 25 mM; E 50, esculetin 50 mM.

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Fig. 5. The esculetin-induced collapse of the MMP was prevented by treatment with CsA in MGC-803 cells. (A) The images of western blot show the effect of esculetin on CypD and ANT. After treatment with esculetin for 24 h, the expression of CypD and ANT was examined by western blot analysis. Data represent similar results from three independent experiments. The images of confocal microscopy (B) and bar graphs (C) show that esculetin induced a disruption of the MMP in a dose-dependent manner in MGC-803 cells. The collapse of the MMP was blocked by pretreatment with CsA, but not z-VAD, BA or NAC. The level of mitochondrial depolarization was observed by confocal microscopy and expressed as the red/green fluorescence intensity ratio. (D) The bar graphs show that esculetin induced the increase of intracellular ROS in a dose-dependent manner. The increase of ROS was prevented by pretreatment with CsA and NAC, but not z-VAD or BA. The level of intracellular ROS was detected by flow cytometry using DCFH-DA fluorescent dye. The cells were preincubated with z-VAD (0.1 mM), CsA (5.0 mM), NAC (1.0 mM) or BA (5.0 mM) for 2 h before the addition of esculetin for 24 h. The data represent the mean ± SD of three independent experiments, *P value represents significant differences between conditions where P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

substantial decrease of the MMP in a dose-dependent manner, as revealed by cell staining with a transmembrane potential-sensitive dye, JC-1 (Fig. 5B and C). We found that the esculetin-induced collapse of the MMP was prevented by treatment with CsA (CsA can specifically interact with CypD (a component of MPTP) and affects pore permeability) [29,30], but not by treatment with BA (an inhibitor of adenine nucleotide translocase (ANT), which is also a component of MPTP) [31] or the antioxidant, NAC (Fig. 5B and C). In addition, we analyzed the production of intracellular ROS in esculetin-treated and untreated cells by flow cytometry. As shown in Fig. 5D, ROS in the esculetin-treated MGC-803 cells exhibited a significant increase in a dose-dependent manner and pretreatment of the cells with CsA or NAC effectively suppressed esculetininduced ROS accumulation. Altogether, these findings demonstrate that esculetin-induced apoptosis in MGC-803 cells is mediated by the mitochondrial pathway, which is associated with ROS. 3.4. Esculetin-induced apoptosis is dependent on CypD and ROS accumulation We investigated the role of ROS and the significance of CypD in mediating esculetin-induced cytotoxicity and apoptosis. The increase of apoptosis and massive cell death, which were observed in the esculetin-treated cells, can be largely prevented by

pretreatment with CsA or NAC. Additionally, BA failed to suppress esculetin-induced cytotoxicity and apoptosis (Fig. 6). These date implied that the increase of cellular ROS was a critical factor underlying esculetin-induced cell death and that CypD might be specific for esculetin-induced cytotoxicity. To further investigate the critical role of CypD in esculetininduced cell death, we used siRNA to knock down the expression of CypD in MGC-803 cells, and then evaluated its effect on esculetin-induced cytotoxicity and apoptosis. As shown in Fig. 7, the cells with CypD siRNA treatment showed a clear suppression of esculetin-induced cell death and apoptosis, confirming the key role of CypD in mediating esculetin-induced cell death. 4. Discussion Chemotherapy drugs, including doxorubicin, 5-fluorouracil, cisplatin, and mitomycin C, have been shown to have great potential to treat gastric cancer [32]. Unfortunately, all of these anticancer drugs not only kill pathologic cells, but also induce an acceleration of the death of normal cells [33]. Therefore, the importance of new chemopreventive and antitumor agents that are more effective but less toxic is becoming increasingly apparent. The use of esculetin has been recommended because of its broad range of activities. The antiproliferative effect of coumarins on gastric

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Fig. 6. Esculetin-induced apoptosis is dependent on CypD and ROS accumulation. (AeC) Bar graphs show that esculetin-induced cell death was blocked by pretreatment with CsA (A) and NAC (C), but not BA (B), in MGC-803 cells. The cell viability was detected by the MTT assay. (DeF) The bar graphs show that esculetin-induced apoptosis was decreased by pretreatment with CsA (D) or NAC (F), but not BA (E), in MGC-803 cells. The cell apoptotic rates were detected by TUNEL assay. The cells were pretreated with CsA (5.0 mM), BA (5.0 mM) or NAC (1.0 mM) for 2 h, and then the cells were incubated with esculetin for 24 h. The data represent the mean ± SD of three independent experiments, *P value represents significant differences between conditions where P < 0.05.

cancer cells has been studied [34]; however, the mechanism by which esculetin prevents human gastric cancer cell proliferation is still largely unknown. In this study, we found that esculetin effectively inhibited cell proliferation in a dose- and time-dependent manner in human gastric cancer SGC-7901, MGC-803 and BGC823 cells. Investigating the mechanism by which human gastric cancer cells undergo a suppression of cellular proliferation in response to esculetin treatment, we found that esculetin induced apoptosis in human gastric cancer cells. Moreover, esculetin treatment increased the activity of caspase-9 and caspase-3 in a dose-dependent manner compared to PBS-treated cells and resulted in the appearance of the 85-kDa PARP cleavage product. Caspase activity and PARP cleavage are intracellular signs of the activation of the apoptotic machinery [35]. In addition, esculetininduced apoptosis as well as cytotoxicity was largely attenuated by the caspase inhibitor, z-VAD. Together, these data demonstrate that esculetin induces cytotoxicity of human gastric cancer cells through an apoptotic mechanism that is dependent upon caspase activation. The apoptotic pathways can be broadly grouped into two main categories: the extrinsic or receptor mediated pathway and the intrinsic or mitochondrial mediated pathway [36]. In the receptor mediated pathway, cell-death signals are conveyed to the cell by means of external signaling molecules, such as TNFa and CD95L [37]; the intrinsic pathway is the main cellular fail-safe mechanism against damage of various types (DNA damage, nucleotide imbalance and hypoxia-induced reactive oxygen species) [37,38]. Caspase-8 and casepase-9 are the apical proteases in the extrinsic and intrinsic pathways, respectively. This study suggests that esculetin-induced apoptosis is mediated through the mitochondrial pathway. Several lines of evidence support this conclusion. First, esculetin treatment increased the activity of caspase-9 and caspase-3 in a dose-dependent manner compared to the vehicle-

treated cells, but caspase-8 activity in the esculetin-treated and vehicle-treated cells remained unaffected. Second, esculetin dosedependently enhanced the expression of Bax and Bak (proapoptotic regulatory proteins), down-regulated the expression of Bcl-2 and Bcl-xL (anti-apoptotic regulatory proteins), and induced cytochrome c release from the mitochondria into the cytoplasm. The fate of the cell is determined at the mitochondrial membrane by the balance between the pro-apoptotic (Bax and Bak) and antiapoptotic (Bcl2 and Bcl-xL) members of the Bcl2 family in the mitochondrial pathway [39,40]. Third, esculetin caused a dramatic decrease of the MMP, which could be blocked by CsA, an agent that prevents the opening of MPTP by binding to CypD. Based on the above analysis, we conclude that the mitochondria play an essential role in esculetin-induced apoptosis. ROS are chemically reactive molecules that are constantly generated and eliminated during diverse biological and cellular reactions, and are known to have a dual role as deleterious and beneficial species [41,42]. Unregulated and redundant ROS are highly toxic to cells because of their peroxidative activity toward biological constituents. Some previous investigations have reported that the generation of ROS is associated with the disruption of the mitochondrial membrane potential [43,44]. Consistent with these findings, we found that intracellular ROS was elevated after treatment with esculetin, which was accompanied by mitochondrial depolarization. In addition, CsA could effectively attenuate the esculetin-induced ROS elevation, which demonstrated that the effects of esculetin on CypD resulted in massive ROS release from the mitochondria and accumulate in cytoplasm. Furthermore, the antioxidant NAC effectively blocked cellular ROS production and prevented esculetinetriggered apoptosis and cell death, suggesting that ROS play a critical role in esculetin-induced cytotoxicity. Our results indicated that an increase of ROS accumulation not only was a consequence of mitochondrial dysfunction, but also triggered

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Fig. 7. Attenuation of esculetin-induced apoptotic cell death by siRNA knockdown of CypD. (A) Images of the western blot show that the expression of CypD was silenced by siRNA in MGC-803 cells. (B) The bar graphs show that the cytotoxicity of esculetin was suppressed by siRNA against CypD. (C) The bar graphs show that esculetin-induced apoptosis was decreased by silencing the expression of CypD in MGC-803 cells. The cell apoptotic rates were detected by a TUNEL assay. The cells were incubated with esculetin for 24 h. (D) The flow cytometry images show that esculetin-induced apoptosis was decreased upon the silencing of CypD expression in MGC-803 cells. The level of apoptosis was determined by the annexin V-FITC/PI double staining assay. The cells were incubated with esculetin for 24 h. The data represent the mean ± SD of three independent experiments, *P value represents significant differences between conditions where P < 0.05.

esculetin-induced apoptosis in MGC-803 cells. The mitochondrial permeability transition pore (MPTP) is a nonspecific pore in the inner mitochondrial membrane, the opening of which is triggered by various stimuli, including mitochondrial depolarization, mitochondria swelling, Ca2þ release, outer mitochondrial membrane rupture, and the release of cell deathassociated proteins (i.e. cytochrome c) to the cytosol [45]. The mitochondrial permeability transition pore is composed of at least three proteins, including the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, the adenine

nucleotide translocator (ANT) in the inner mitochondrial membrane, and CypD in the matrix [29,46]. Under the resting condition, CypD resides in the mitochondrial matrix to keep the MPTP shut [46,47]; however, it associates with ANT in the inner mitochondrial membrane to open the MPTP when facing critical conditions [48]. Our results indicated that CsA could effectively attenuate esculetininduced apoptosis, suggesting that the cytotoxicity of esculetin was closely associated with CypD, which seemed to play an important role in esculetin-induced cell death. This finding was validated by the silencing of CypD. Moreover, the observation that BA failed to

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attenuate esculetin-induced apoptosis or cell death further suggests that CypD might be specific for esculetin-induced cytotoxicity. There is an interesting result in our study, the esculetin-induced collapse of MMP could not be prevented by NAC, how could the esculetin-induced apoptosis being prevented by NAC? Next, we try to explain this conflict result. MMP can be regulated by many factors, including the change of membrane permeability, the expression level of Bcl-2 family, intracellular oxidative stress [49e52]. Mitochondria are the major source of ROS, and ROS generation is mediated by permeability transition [53]. It has been reported that mitochondrial derived Ca2þ could regulate MPTP opening and quantal bursting mode of ROS production in many types of cells [54]. Some studies have also showed that ROS could induce the collapse of MMP, and NAC significantly blocked the loss of MMP [49]. However, in this study, we have proved that esculetin-induced collapse of MMP was associated with the opening of MPTP and the expression level of the Bcl-2 family. Role of ROS in triggering apoptosis is not entirely understood [55]. It has been reported that excessive ROS accumulation can lead to cellular injury, including lipid peroxidation, protein oxidation, enzyme inactivation, and oxidative DNA damage, and eventually lead to cell apoptosis or necrosis [56]. In addition, some previous investigations have reported that oxidation of the mitochondrial pores by ROS may contribute to cytochrome c release, resulting in autoactivation of initiator caspases [57]. However, to determine whether esculetin induced-ROS trigger apoptosis was dependent upon the above pathway, further investigation need be performed, the results should contribute to the additional understanding of the cell death mechanism. In conclusion, our results demonstrate that esculetin induced apoptosis via the caspase-dependent mitochondrial pathway in human gastric cancer cells, in which CypD mediated the cytotoxic action by triggering the opening of the mitochondrial permeability transition pore. The generation of ROS is necessary for esculetininduced apoptosis. These results reveal a novel mechanism of esculetin on gastric cancer cells and suggest that esculetin could be a novel agent in the treatment of gastric cancer; however, the relationship between ROS and CypD requires further investigations. In addition, an investigation of esculetin in mouse models should be performed. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments We thank the highly qualified native English-speaking editors (Elsevier's WebShop) for editing work. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2015.09.015. References [1] J. Ferlay, H.R. Shin, F. Bray, D. Forman, C. Mathers, D.M. Parkin, Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008, Int. J. Cancer 127 (12) (2010) 2893e2917. [2] D.M. Parkin, F. Bray, J. Ferlay, P. Pisani, Global cancer statistics, 2002, CA Cancer J. Clin. 55 (2) (2005) 74e108. [3] J.A. Ajani, Evolving chemotherapy for advanced gastric cancer, Oncologist 10 (Suppl. 3) (2005) 49e58. [4] F. De Vita, F. Giuliani, G. Galizia, C. Belli, G. Aurilio, G. Santabarbara, F. Ciardiello, G. Catalano, M. Orditura, Neo-adjuvant and adjuvant

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