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Q6 Q5
Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells Ah Young Lee a, 1, Young-Ah Han a, 1, Ji-Eun Kim a, Seong-Ho Hong a, Eun-Jung Park b, Myung-Haing Cho a, c, d, e, f, * a Laboratory of Toxicology, BK21 PLUS Program for Creative Veterinary Science Research, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul National, University, Seoul 151-742, Republic of Korea b Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea c Graduate School of Convergence Science and Technology, Seoul National University, Suwon 443-270, Republic of Korea d Graduate Group of Tumor Biology, Seoul National University, Seoul 151-742, Republic of Korea e Advanced Institute of Convergence Technology, Seoul National University, Suwon 443-270, Republic of Korea f Institute of GreenBio Science Technology, Seoul National University, Pyeongchang 232-916, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 January 2015 Received in revised form 1 May 2015 Accepted 9 May 2015 Available online xxx
In this study, we examined the mechanism underlying the effect of Saururus chinensis Baill (saururaceae) on hepatocellular carcinoma HepG2 cells. HepG2 cells and Chang cells were exposed to various concentrations of S. chinensis Baill extract (SC-E) for 24 h. SC-E affected more significantly HepG2 cells than Chang cells in terms of cell viability and ATP production. Therefore, current study examined detailed mechanism how SC-E affected HepG2 cell survival. We found that SC-E (75 and 150 mg/ml) induced apoptosis via oxidative stress. SC-E also caused CCAAT-enhancer-binding protein homologous protein (CHOP) activation by dissociating the binding immunoglobulin protein (BiP) from inositol-requiring 1a (IRE1a) in the endoplasmic reticulum (ER) and induced Bax, cytochrome c release to cytosol, caspase-3 activation, and poly ADP ribose polymerase (PARP) cleavage, resulting in HepG2 cell apoptosis. Furthermore, SC-E caused ER Ca2þ leakage into the cytosol; ER dilation and mitochondrial membrane damage were observed in transmission electron microscopy (TEM). Taken together, our results demonstrated that SC-E induced cancer cell apoptosis specifically through ER stress. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Saururus Chinesis Baill ER stress Apoptosis Anticancer agent Natural product
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common cancer worldwide. It is the second most serious reason of cancer death in men, and it is the sixth leading cause of cancer death. An estimated 748,300 new HCC cases and 695,900 cancer deaths happened worldwide (Jemal et al., 2011). The increase in cancer incidence and death rates necessitates the identification of drug candidate for cancer prevention or treatment. Searches for anticancer agent from natural sources have been increased because of the toxicity and adverse effect of synthetic anticancer drugs. Recent studies have reported growth inhibition or cell death in human
* Corresponding author. Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea. E-mail address: [email protected] (M.-H. Cho). 1 Equal contribution to this work.
cancer cells on exposure to extracts or constituents of natural products (Tan et al., 2011). Saururus chinensis Baill (saururaceae), a perennial herbal plant, cultivated in China and Korea has traditionally been used to treat inflammation in diverse condition such as edema, gonorrhea, and asthma (Yoo et al., 2008; Yu et al., 2008; Chang et al., 2011). Several studies have reported that the ethanol extract of S. chinensis Baill (SC-E) decreased inflammation by inhibiting intracellular nitric oxide, prostaglandin E2, and various inflammatory cytokines released by lipopolysaccharide stimulation of raw 264.7 macrophages (Kim et al., 2003; Yoo et al., 2008; Li et al., 2014). SC-E also regulated blood lipid level in animal model and suppressed the activity of a-glucosidase for the anti-diabetic effect (Hwang et al., 2007; Yu et al., 2008). The endoplasmic reticulum (ER) has been found to be a central player in many apoptotic pathways. Many studies have shown that apoptotic cell death is closely related to ER stress. The ER plays an
http://dx.doi.org/10.1016/j.fct.2015.05.008 0278-6915/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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essential role in maintaining intracellular protein synthesis, Ca2þ homeostasis, protein folding, and controlling cell homeostasis (Yoshida, 2007). However, some physiological and pathological conditions, such as hypoxia, oxidative stress, viral infection and Ca2þ depletion in the ER can impair the balance between the protein folding load and capacity of the ER. CCAAT-enhancer-binding protein homologous protein (CHOP) also affects as a transcription factor regulating the genes related to cell survival or death. The expression of CHOP as an important transcription factor associated in early cell death is under the signal control of dsRNA-activated activating transcription factor 6 (ATF6), protein kinase-like ER kinase (PERK) and inositol-requiring 1a (IRE1a) (Oyadomari and Mori, 2004; Cao et al., 2013). In fact, recent line of evidences reported that ER stress is closely associated with apoptosis (Yoshida, 2007; Heath-Engel et al., 2008). Apoptosis, a type of programmed cell death, is a physiological process whereby cells die as a result of exposure to abnormal condition. Apoptotic cells proceed through cell condensation, nuclear fragmentation, and chromatin condensation. Various stress stimuli such as hypoxia, oxidative stress, and Ca2þ ion or toxic irritation induce DNA damage followed by increase of membrane permeability and finally the release of cytochrome c into cytosol, which activates apoptosome formation followed by activation of caspase-9, which in turn induces apoptosis by activating of caspase3 (Heath-Engel et al., 2008). Many studies have reported that different apoptotic cell death plays a novel role with important implications in HCC cells, therefore, chemotherapeutic drugs through apoptotic cell death could be a nice candidate for HCC treatment (Kern et al., 2006; Zuo et al., 2015). Our preliminary study indicated that SC-E might cause toxic effects on cancer cells through ER stress. Therefore, we hypothesized that SC-E might induce apoptosis through ER stress in the HepG2 cells. We report here that SC-E can induce ER stressmediated apoptosis in HepG2 cells, indicating that SC-E may serve as a natural antitumor agent.
Cell media were removed and treated with serial dilutions of SC-E and incubated for 24 h. Then, the cell media were removed, 10 ml CCK-8 solution was added, and the cells were incubated for another 1 h. The absorbance was measured with an ELISA microplate reader (Bio-Rad, Richimond, CA, USA) at 450 nm. Cell viability was calculated as follows: % cell viability ¼ (absorbance of treated cells/ absorbance average of untreated cells; control cells) 100. 2.3. Measurement of ATP production To examine the energy metabolism of cells, ATP level was analyzed using the CellTiter-Glo luminescent cell viability assay kit (Promega, Madison, WI, USA). The cells were seeded on the whitewalled 96-well optical bottom plate at 5 103 cells/well and incubated for 24 h. Then, serial dilutions of SC-E were added, followed by incubation for another 24 h. All the wells were treated with 50 ml of CellTiter-Glo reagent for 1 min in a shaking incubator at 60 rpm with protective lights. The luminescence was measured by luminometer (Berthold, Bad Wildbad, Germany). 2.4. Western blot assay The total cell lysate was separated on SDS-PAGE gel, and the proteins were transferred using Life Technologies iBlot system (Life Technologies). The membranes were blocked for 1 h at room temperature in Tris-buffered saline Tween (TTBS) with 5% skim milk and incubated overnight at 4 C with appropriate primary antibodies such as BiP, IRE1a, CHOP, PARP, Calnexin (Cell Signaling, Danvers, MA, USA) or Bcl-xL and Bax (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a-tubulin (AbFrontier, Seoul, Korea) and washed 3 times in TTBS. Then, the membranes were incubated with a secondary antibody (Life Technologies) for overnight at 4 C, and washed 3 times in TTBS. Quantification of the proteins assayed was performed using the CS analyzer 3.0 program (ATTO Corp., Tokyo, Japan).
2. Materials and methods 2.1. Cell culture and treatment
2.5. Measurement of GSH/GSSG
Chang (normal liver cell line) cells were kindly provided by Dr. EJ Park (Ajou University, Suwon, Korea) and were maintained in highglucose Dulbecco's modified Eagle's medium (DMEM, GE Health Care Science, Pittsburgh, PA, USA). HepG2 (human hepatocellular liver carcinoma cell line) cells were obtained from Korea Cell Line Bank (KCLB, Seoul, Korea) and were maintained in Minimum Essential Media (MEM, Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (Life Technologies) and 1% penicillinestreptomycin (Life Technologies) at 37 C in a 5% CO2 incubator. The SC-E, obtained from Jeju Technopark (JTP, Jeju, Korea), was dissolved in ethanol to obtain a stock solution of 10 mg/ ml. The stock solution was used for dilution to required concentrations. For this experiment, the cells were treated with two concentrations (75 and 150 mg/ml) of SC-E for 24 h. Vehicle control cells were treated with vehicle (1.05% ethanol) and positive control cells were treated with thapsigargin (TG, 0.1 mM, SigmaeAldrich, St Louis, MO, USA) for 24 h. ER stress inhibitors such as tauroursodeoxycholic acid (TUDCA) and sodium phenylbutyrate (4PBA) were purchased from SigmaeAldrich.
The GSH/GSSG-Glo™ Assay (Promega) is a luminescence-based system to measure GSH/GSSG ratio. After 24 h of SC-E, and TG treatment, GSH/GSSG ratio was measured using luminometer (Berthold, Bad Wildbad, Germany).
2.2. Cell viability assay Cell viability was measured using Cell Counting Kit (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan). Chang cells and HepG2 cells were seeded in 96-well plates at 5 103 cells/well.
2.6. Apoptotic cell analysis (Annexin-V/PI analysis) Annexin V/PI assay (Koma Biotech, Seoul, Korea) detects apoptotic cells using flow cytometry. After treatment of SC-E (75 mg/ml and 150 mg/ml) and TG (0.1 mM), fluorescence intensity in the cells was measured using FACS AriaII flow cytometer (BD, Franklin Lakes, NJ, USA). 2.7. Caspase-3, 7 activity Caspase-3, 7 activity was analyzed using Apo-ONE® Homogeneous Caspase-3, 7 (Promega). The cells were seeded on the blackwalled 96-well optical bottom plate at 5 103 cells/well and incubated for 24 h. Then, 100 ml of various concentrations of SC-E were added, followed by incubation for another 24 h. All the wells were treated with 50 ml of Apo-ONE® Homogeneous Caspase3, 7 buffer for 4 h at 37 C with protective lights. The fluorescence was measured with Multiple Plate Reader victor 3 (PerkinElmer, Waltham, MA, USA).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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2.8. Monitoring signal of Ca2þ (Fluo-4, AM staining) HepG2 cells were grown on a chamber slide (Thermo Fisher Scientific Inc., Waltham, MA, USA) to 40e50 % confluence. After the cells were incubated for 24 h with treatment, they were washed with phosphate-buffered saline (PBS) and stained with 4 mM Fluo4, AM (Life Technologies) for 1 h at 37 C. Then, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Subsequently, the cells were washed and mounted with mounting media and coverslip. The image was obtained by confocal laser scanning microscopy (LSM710, Carl Zeiss Microscopy GmbH, Oberkochen, Germany).
2.9. Transmission electron microscopy (TEM) analysis Cells were incubated with SC-E or TG for 24 h and washed with PBS. Then, they were fixed with Karnovsky's solution for 2 h at 4 C and washed 3 times for 10 min each with 0.05 M sodium cacodylate buffer. Next, the cells were fixed with a mixture of equal parts of 2% osmium tetroxide and 0.1 M cacodylate buffer for 2 h at 4 C and washed twice with distilled water. They were then stained with 0.5% uranyl acetate overnight at 4 C. The stained cells were subjected to dehydration with ethanol series (30%, 50%, 70%, 80%, 90%, and 100%). The dehydrated cells were embedded overnight in a mixture of equal parts of propylene oxide and Spurr's resin at room temperature. Then, the cells were embedded another overnight in
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Spurr's resin at 70 C. Ultrathin sections of the samples were cut and mounted on copper grids. The sections were stained with lead citrate and uranyl acetate and observed by TEM. All reagents were purchased from Electron Microscopy Sciences (EMS, Hatfield, PA, USA). 2.10. Measurement of the cytochrome c release The cells were seeded on 8 well-chamber slide at 7 103 cells/ well and incubated for 24 h. After the cells were incubated for 24 h with treatment, the cells were stained with Mito Tracker (Life Technologies) for 30 min at 37 C. The slides were blocked for 1 h at room temperature in Tris-buffered saline Tween (TTBS) with 3% BSA and incubated overnight at 4 C with a primary antibody, using cytochrome c (Santa Cruz Biotechnology) and washed 3 times in TTBS. Then, the slides were incubated with a secondary antibody, Alexa Fluor 488 goat anti mouse IgG (Life Technologies) for 1 h at room temperature, and washed 3 times in TTBS. The image was obtained by LSM710 (Carl Zeiss Microscopy GmbH). 2.11. Statistical analysis All data have been expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed by comparing the vehicle control and treated groups using Student's t-test. P < 0.05 was considered statistically significant.
Fig. 1. Cytotoxic effects of SC-E on the Chang and HepG2 cell lines. Chang (A, B) and HepG2 (C, D) cells were treated with diverse concentrations of SC-E (18.8e300 mg/ml for 24 h). Cell viability was measured by the CCK-8 assay. The reduction of ATP production by SC-E (18.8e300 mg/ml for 24 h) was detected in Chang (B) and HepG2 cells (D). Data are expressed as mean ± standard error of the mean (SEM). *Significantly different from control (p < 0.05), **Significantly different from control (p < 0.01), ***Significantly different from control (p < 0.001).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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Fig. 2. Generation of intracellular ROS and apoptotic cells in response to SC-E. Generation of intracellular ROS was detected by GSH/GSSG ratio (A) and apoptosis was measured by Annexin V/PI (BeD). Apoptotic cells were detected by FACS analysis (B). % of early apoptotic cells were Annexin-V positive and PI negative (C). % of apoptotic cells were Annexin-V positive, PI negative and Annexin-V positive, PI positive (D). Data are expressed as mean ± SEM. *Significantly different from vehicle control (p < 0.05), **Significantly different from Q7 vehicle control (p < 0.01), ***Significantly different from vehicle control (p < 0.001). Compared with vehicle control (*), control (#), SC-E 75 mg/ml ($) and thapsigargin (&).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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3. Results
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E treatment, more severe cytotoxicity was observed in HepG2 cells compared to Chang cells (Fig. 1A and C).
3.1. Cytotoxicity in Chang cells and HepG2 cells 3.2. ATP production in Chang cells and HepG2 cells MTT assay was performed to investigate the effect of SC-E on cell viability in Chang cells and HepG2 cells following incubation with various concentrations (18.8e300 mg/ml) of SC-E for 24 h. After SC-
ATP production was assessed after SC-E treatment to examine the effect of SC-E on mitochondrial function in HepG2 and Chang
Fig. 3. Effects of SC-E on ER stress-associated proteins and apoptosis-associated proteins in HepG2 cells. ER stress-and apoptosis-related proteins were analyzed by Western blot assay. Cells were treated with SC-E (75 and 150 mg/ml) or TG (0.1 mM) for 24 h. Each protein was analyzed using specific antibodies (A and D). The average band thickness was calculated (B and E). Caspase-3, 7 activity was increased by SC-E treatment in a concentration-dependent manner (C). Data are expressed as mean ± SEM. a-tubulin was used as a protein loading control and for band density normalization. *Significantly different from vehicle control (p < 0.05), **Significantly different from vehicle control (p < 0.01), ***Significantly different from vehicle control (p < 0.001). Compared with vehicle control (*), control (#), SC-E 75 mg/ml ($) and thapsigargin (&).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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Fig. 3. (continued).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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cells. ATP production was decreased concentration-dependently in both Chang cells and HepG2 cells (Fig. 1B and D). SC-E decreased ATP production in HepG2 cells more significantly compared with Chang cells. These results indicated that SC-E affected more severely on cell viability and ATP production in HepG2 cells than those in Chang cells. Taken together with cytotoxicity results, HepG2 cells were chosen for current study and SC-E concentration was set at 75 mg/ml and 150 mg/ml because these concentrations showed meaningful cytotoxicity in HepG2 cells. 3.3. SC-E induced oxidative stress in HepG2 cells As explained, luminescence-based GSH/GSSG-Glo™ assay was performed in order to evaluate the cellular redox status of HepG2 cells after SC-E treatment. The ratio was decreased as a function of SC-E concentration and TG (Fig. 2A). Since the ROS level and GSH/ GSSG ratio reflect the oxidative stress in cells (Lin et al., 2014) in conjunction of finding that GSH/GSSG ratio is decreased under oxidative stress (Zitka et al., 2012), SC-E seemed to affect HepG2 cell viability through oxidative stress. 3.4. SC-E induced apoptosis in HepG2 cells To determine the rate of apoptotic cell death in HepG2 cells, Annexin V/PI assay was performed after treatment with SC-E and TG (Fig. 2B). Early-stage apoptotic cells were Annexin V positive, and PI negative. Late-stage apoptotic cells were Annexin V positive, and PI positive. The rate of apoptotic cell death in the cells was increased significantly by SC-E and TG (Fig. 2C and D). 3.5. SC-E induced apoptosis through ER stress Western blot assay was performed to determine the protein level of ER stress and apoptosis. We found that the protein expression level of ER stress and apoptosis was increased in HepG2 cells after SC-E treatment in a concentration-dependent manner (Fig. 3A and D). These results were reconfirmed by densitometric
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analysis (Fig. 3B and E). SC-E increased ER stress-related proteins expression. SC-E also increased Bax protein expression and PARP cleavage concentration-dependently. However, anti-apoptotic protein Bcl-xL was decreased (Fig. 3D and E). Caspase-3, 7 activity was increased by SC-E treatment in a concentration-dependent manner (Fig. 3C). Release of cytochrome c from mitochondria induced mitochondria-mediated apoptosis, thus, known to cause early cell death (Boris et al., 1998). SC-E induced the release of cy- Q1 tochrome c from mitochondria to cytosol (Fig. S1). Taken altogether, our data indicate that SC-E induces mitochondria-mediated apoptosis in conjunction with ER stress. 3.6. Suppression of endoplasmic reticulum stress reduced apoptosis induced by SC-E Western blot assay was performed to determine whether SC-E affected ER stress-related proteins or apoptosis-related proteins. When the cells were pretreated with 4-PBA, TUDCA as ER stress inhibitors, apoptosis induced by SC-E was reduced significantly (Fig. 4A and B). These ER stress-mediated effects on apoptosis were reconfirmed by caspase-3, 7 activity assay (Fig. 4C). In addition, increase of intracellular free calcium level induced by SC-E was significantly reduced by 4-PBA and TUDCA (Fig. 5). These results support that SC-E induces apoptosis through ER stress. 3.7. SC-E elevated intracellular free Ca2þ Intracellular calcium level is increased by the release of Ca2þ from the ER to the cytosol in the circumstances of cell damage or ER stress. To measure intracellular Ca2þ level, HepG2 cells was exposed to SC-E (75 and 150 mg/ml), TG (0.1 mM) and co-treatment with 4PBA, TUDCA. Then, the cells were stained with Fluo-4, AM. SC-E increased Fluo-4, AM fluorescence (Green) in a concentrationdependent manner compared to untreated control cells. However, co-treatment 4-PBA and TUDCA decreased Fluo-4, AM fluorescence (Fig. 5).
Fig. 4. Effects of ER stress inhibitor, 4-PBA and TUDCA on levels of ER stress-related proteins and apoptosis-related proteins in SC-E treated cells. ER stress-related proteins and apoptosis-related proteins were analyzed by Western blot assay. Cells were treated with SC-E (150 mg/ml) or TG (0.1 mM) and co-treatment ER stress inhibitors: 4-PBA (10 mM) and TUDCA (5 mM). Each protein was analyzed using specific antibodies (A and B). Caspase-3, 7 activity was decreased by co-treatment with 4-PBA and TUDCA (C). Data are expressed as mean ± SEM. *Significantly different from SC-E 150 mg/ml (p < 0.05), **Significantly different from thapsigargin (p < 0.01).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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Fig. 5. Microscopic fluorescence imaging of Fluo-4, AM staining for intracellular Ca2þ level after SC-E treatment. HepG2 cells were treated SC-E (75 and 150 mg/ml), TG (0.1 mM) and co-treatment ER stress inhibitors, 4-PBA (10 mM) and TUDCA (5 mM), and then cells stained with Fluo-4, AM (Green). Scale bar ¼ 10 mm.
3.8. SC-E damaged ER and mitochondrial morphology Extracellular stimuli cause organelle morphology changes that lead to cell dysfunction. TEM analysis was performed to examine whether SC-E treatment caused morphological changes in HepG2
cells. The TEM image distinctly showed morphological changes, such as ER dilation and mitochondrial crista damage at 75 mg/ml SC-E and critical ER swelling and mitochondrial crista and outer membrane damage at 150 mg/ml SC-E, compared to untreated control cells (Fig. 6).
Fig. 6. TEM analysis of the cell response to SC-E. Transmission electron microscopy of unexposed (control) cells (A), and of cells at 24 h after treatment with SC-E 75 mg/ml (B), SC-E 150 mg/ml (C) and TG 0.1 mM (D).N; nuclear, M; mitochondria, ER; endoplasmic reticulum, G; Golgi apparatus. Scale marker indicates 2 mm and 0.5 mm.
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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4. Discussion Recently, cancer cell-specific cell death has been proposed as a new targeted treatment for anticancer therapy (Liu et al., 2014). There is a long history of anticancer drugs identified from herbal medicines, and naturally derived compounds can be a source of powerful anticancer agents. Some studies have reported the inhibition of melanoma formation and cell death in prostate and breast cancer cells using S. chinensis Baill constituents. The compound manassantin A isolated from SC-E has anti-proliferative effect in different cancer cell lines (Rao and Reddy, 1990; Feng et al., 2011; Lee et al., 2012). However, there were no studies on ER stressmediated apoptosis regarding SC-E (Kim et al., 2011; Lee et al., 2013). This is why current study has focused on the function of SC-E as a potential effective anticancer reagent through ER stressmediated apoptosis. Many lines of evidences have shown that ROS production is an important factor for apoptosis induction. ROS, which is continually generated and eliminated in biological systems, plays a role in diverse biological functions in the normal state but also induces pathological progression in abnormal state (Yang et al., 2014). Imbalance between generation and elimination leads to overproduction of ROS, which could induce apoptosis by cell death signaling (Li et al., 2015). Other studies have mentioned the role of ROS in caspase activation and ER stress during apoptosis (Pierre et al., 2013; Tagawa et al., 2008). Our current results also showed the evidence that oxidative stress was associated with ER stress in SC-E treated cells. ER stress is well known as a crucial part of the regulation of cellular homeostasis, especially in the unfolded protein response (UPR) (Chen et al., 2014). Binding immunoglobulin protein (BiP), also called GRP78, is a glucose-regulated protein and is one of the most efficient ER luminal chaperone proteins. BiP is associated with three ER transmembrane proteins, IRE1a, PERK, and ATF6, during unstressed conditions (Dara et al., 2011; Wang et al., 2014). Under ER stress, BiP is activated, UPR signal is initiated, and the three ER transmembrane proteins are dissociated from BiP. Activated IRE1a produces spliced XBP1 by splitting XBP fragments and activates proapoptotic proteins through activation of the signaling cascade controlling cell survival or death. CHOP is a member of C/EBP family and known to be induced highly under ER stress (Fig. 3A and B). The activation of IRE1a, PERK and ATF6 has been reported to increase CHOP protein expression, activating and initiating the ER stressinduced apoptotic cascade (Qu et al., 2013; Chen et al., 2014; Liu et al., 2014; Yang et al., 2014). We observed that the protein expression levels of IRE1a and CHOP were increased after treatment with SC-E in HepG2 cells. Our data suggested that SC-E treatment induced ER stress and apoptosis. Our results also demonstrated that the increase of cytosolic Ca2þ concentration was associated with ER stress (Fig. 5). In fact, recent works have suggested that ER calcium depletion by TG probably triggers apoptosis while overexpression of the anti-apoptotic Bcl-2 protein, which anchors the intracellular membranes, maintains the Ca2þ homeostasis in ER, thereby preventing apoptosis induction by TG (Yoshida et al., 2006). Moreover, the release of Ca2þ from the ER has known to play an important relation between the ER and the major mechanism in controlling apoptosis (Li et al., 2012a,b; Cao et al., 2013). Then, the influx of Ca2þ to the mitochondria activates the release of cytochrome c that causes apoptosis. All things taken together, our results showed that SC-E mediated the increase of cytosolic Ca2þ concentration through ER stress. Caspases have been reported to play an essential role in apoptosis or scheduled cell death. The release of cytochrome c to the cytosol in apoptosis along the mitochondrial pathway is related to activation of downstream caspases, especially caspase-3, which
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are crucial in DNA fragmentation associated with morphological changes and apoptosis of mitochondria (Kim et al., 2009; Li et al., 2012a,b). Furthermore, caspase-3 is necessary for PARP cleavage, an important marker of apoptosis. Both cytochrome c-related apoptosis and cytochrome c release are mainly controlled by members of the Bcl-2 family. Bax and Bcl-2 are recognized as important regulators. Bax inserts into the mitochondrial outer membrane and translocates to the mitochondria to facilitate the release of cytochrome c (Huang et al., 2006). Although the association of Bcl-2 and Bax neutralizes the proapoptotic effects, a large increase in Bax expression induces apoptosis. Our results demonstrated that SC-E was an inducer of apoptosis in HepG2 cells. SC-Einduced apoptosis was dependent on the activation of mitochondria-mediated caspases and on the interaction between Bax and the mitochondria (Fig. 3D and E). In conclusion, ER stress-mediated apoptosis induced by SC-E could inhibit the survival of hepatocellular carcinoma HepG2 cells. Our results demonstrated that SC-E induced ER stress in HepG2 cells, disrupted intracellular calcium homeostasis, and led to cell death through apoptosis by mitochondrial damage. We propose that SC-E could be used as an anticancer agent for liver cancer. Acknowledgments This work was supported by the Bio-Synergy Research Project (NRF-2012M3A9C4048819) of the Ministry of Science, ICT and Future Planning through the National Research Foundation. CLSM images were acquired at National Center for Inter-University Research Facilities in Seoul National University. This study was supported through BK21 PLUS Program for Creative Veterinary Science Research. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fct.2015.05.008. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2015.05.008. Uncited references Zhivotovsky et al., 1998. References Cao, A., Li, Q., Yin, P., Dong, Y., Shi, H., Wang, L., Ji, G., Xie, J., Wu, D., 2013. Curcumin induces apoptosis in human gastric carcinoma AGS cells and colon carcinoma HT-29 cells through mitochondrial dysfunction and endoplasmic reticulum stress. Apoptosis 18 (11), 1391e1402. Chang, J.S., Lee, S.W., Kim, M.S., Yun, B.R., Park, M.H., Lee, S.G., Park, S.J., Lee, W.S., Rho, M.C., 2011. Manassantin A and B from Saururus chinensis inhibit interleukin-6-induced signal transducer and activator of transcription 3 activation in Hep3B cells. J. Pharmacol. Sci. 115 (1), 84e88. Chen, Y., Gui, D., Chen, J., He, D., Luo, Y., Wang, N., 2014. Down-regulation of PERKATF4-CHOP pathway by Astragaloside IV is associated with the inhibition of endoplasmic reticulum stress-induced podocyte apoptosis in diabetic rats. Cell. Physiol. Biochem. 33 (6), 1975e1987. Dara, L., Ji, C., Kaplowitz, N., 2011. The contribution of endoplasmic reticulum stress to liver diseases. Hepatology 53 (5), 1752e1763. Feng, Y., Wang, N., Zhu, M., Feng, Y., Li, H., Tsao, S., 2011. Recent progress on anticancer candidates in patents of herbal medicinal products. Recent Pat. Food Nutr. Agric. 3 (1), 30e48. Heath-Engel, H.M., Chang, N.C., Shore, G.C., 2008. The endoplasmic reticulum in apoptosis and autophagy: role of the BCL-2 protein family. Oncogene 27 (50), 6419e6433. Huang, Y.T., Huang, D.M., Chueh, S.C., Teng, C.M., Guh, J.H., 2006. Alisol B acetate, a triterpene from Alismatis rhizoma, induces Bax nuclear translocation and
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells Ah Young Lee a, 1, Young-Ah Han a, 1, Ji-Eun Kim a, Seong-Ho Hong a, Eun-Jung Park b, Myung-Haing Cho a, c, d, e, f, * a Laboratory of Toxicology, BK21 PLUS Program for Creative Veterinary Science Research, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul National, University, Seoul 151-742, Republic of Korea b Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea c Graduate School of Convergence Science and Technology, Seoul National University, Suwon 443-270, Republic of Korea d Graduate Group of Tumor Biology, Seoul National University, Seoul 151-742, Republic of Korea e Advanced Institute of Convergence Technology, Seoul National University, Suwon 443-270, Republic of Korea f Institute of GreenBio Science Technology, Seoul National University, Pyeongchang 232-916, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 January 2015 Received in revised form 1 May 2015 Accepted 9 May 2015 Available online xxx
In this study, we examined the mechanism underlying the effect of Saururus chinensis Baill (saururaceae) on hepatocellular carcinoma HepG2 cells. HepG2 cells and Chang cells were exposed to various concentrations of S. chinensis Baill extract (SC-E) for 24 h. SC-E affected more significantly HepG2 cells than Chang cells in terms of cell viability and ATP production. Therefore, current study examined detailed mechanism how SC-E affected HepG2 cell survival. We found that SC-E (75 and 150 mg/ml) induced apoptosis via oxidative stress. SC-E also caused CCAAT-enhancer-binding protein homologous protein (CHOP) activation by dissociating the binding immunoglobulin protein (BiP) from inositol-requiring 1a (IRE1a) in the endoplasmic reticulum (ER) and induced Bax, cytochrome c release to cytosol, caspase-3 activation, and poly ADP ribose polymerase (PARP) cleavage, resulting in HepG2 cell apoptosis. Furthermore, SC-E caused ER Ca2þ leakage into the cytosol; ER dilation and mitochondrial membrane damage were observed in transmission electron microscopy (TEM). Taken together, our results demonstrated that SC-E induced cancer cell apoptosis specifically through ER stress. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Saururus Chinesis Baill ER stress Apoptosis Anticancer agent Natural product
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common cancer worldwide. It is the second most serious reason of cancer death in men, and it is the sixth leading cause of cancer death. An estimated 748,300 new HCC cases and 695,900 cancer deaths happened worldwide (Jemal et al., 2011). The increase in cancer incidence and death rates necessitates the identification of drug candidate for cancer prevention or treatment. Searches for anticancer agent from natural sources have been increased because of the toxicity and adverse effect of synthetic anticancer drugs. Recent studies have reported growth inhibition or cell death in human
* Corresponding author. Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea. E-mail address: [email protected] (M.-H. Cho). 1 Equal contribution to this work.
cancer cells on exposure to extracts or constituents of natural products (Tan et al., 2011). Saururus chinensis Baill (saururaceae), a perennial herbal plant, cultivated in China and Korea has traditionally been used to treat inflammation in diverse condition such as edema, gonorrhea, and asthma (Yoo et al., 2008; Yu et al., 2008; Chang et al., 2011). Several studies have reported that the ethanol extract of S. chinensis Baill (SC-E) decreased inflammation by inhibiting intracellular nitric oxide, prostaglandin E2, and various inflammatory cytokines released by lipopolysaccharide stimulation of raw 264.7 macrophages (Kim et al., 2003; Yoo et al., 2008; Li et al., 2014). SC-E also regulated blood lipid level in animal model and suppressed the activity of a-glucosidase for the anti-diabetic effect (Hwang et al., 2007; Yu et al., 2008). The endoplasmic reticulum (ER) has been found to be a central player in many apoptotic pathways. Many studies have shown that apoptotic cell death is closely related to ER stress. The ER plays an
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Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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essential role in maintaining intracellular protein synthesis, Ca2þ homeostasis, protein folding, and controlling cell homeostasis (Yoshida, 2007). However, some physiological and pathological conditions, such as hypoxia, oxidative stress, viral infection and Ca2þ depletion in the ER can impair the balance between the protein folding load and capacity of the ER. CCAAT-enhancer-binding protein homologous protein (CHOP) also affects as a transcription factor regulating the genes related to cell survival or death. The expression of CHOP as an important transcription factor associated in early cell death is under the signal control of dsRNA-activated activating transcription factor 6 (ATF6), protein kinase-like ER kinase (PERK) and inositol-requiring 1a (IRE1a) (Oyadomari and Mori, 2004; Cao et al., 2013). In fact, recent line of evidences reported that ER stress is closely associated with apoptosis (Yoshida, 2007; Heath-Engel et al., 2008). Apoptosis, a type of programmed cell death, is a physiological process whereby cells die as a result of exposure to abnormal condition. Apoptotic cells proceed through cell condensation, nuclear fragmentation, and chromatin condensation. Various stress stimuli such as hypoxia, oxidative stress, and Ca2þ ion or toxic irritation induce DNA damage followed by increase of membrane permeability and finally the release of cytochrome c into cytosol, which activates apoptosome formation followed by activation of caspase-9, which in turn induces apoptosis by activating of caspase3 (Heath-Engel et al., 2008). Many studies have reported that different apoptotic cell death plays a novel role with important implications in HCC cells, therefore, chemotherapeutic drugs through apoptotic cell death could be a nice candidate for HCC treatment (Kern et al., 2006; Zuo et al., 2015). Our preliminary study indicated that SC-E might cause toxic effects on cancer cells through ER stress. Therefore, we hypothesized that SC-E might induce apoptosis through ER stress in the HepG2 cells. We report here that SC-E can induce ER stressmediated apoptosis in HepG2 cells, indicating that SC-E may serve as a natural antitumor agent.
Cell media were removed and treated with serial dilutions of SC-E and incubated for 24 h. Then, the cell media were removed, 10 ml CCK-8 solution was added, and the cells were incubated for another 1 h. The absorbance was measured with an ELISA microplate reader (Bio-Rad, Richimond, CA, USA) at 450 nm. Cell viability was calculated as follows: % cell viability ¼ (absorbance of treated cells/ absorbance average of untreated cells; control cells) 100. 2.3. Measurement of ATP production To examine the energy metabolism of cells, ATP level was analyzed using the CellTiter-Glo luminescent cell viability assay kit (Promega, Madison, WI, USA). The cells were seeded on the whitewalled 96-well optical bottom plate at 5 103 cells/well and incubated for 24 h. Then, serial dilutions of SC-E were added, followed by incubation for another 24 h. All the wells were treated with 50 ml of CellTiter-Glo reagent for 1 min in a shaking incubator at 60 rpm with protective lights. The luminescence was measured by luminometer (Berthold, Bad Wildbad, Germany). 2.4. Western blot assay The total cell lysate was separated on SDS-PAGE gel, and the proteins were transferred using Life Technologies iBlot system (Life Technologies). The membranes were blocked for 1 h at room temperature in Tris-buffered saline Tween (TTBS) with 5% skim milk and incubated overnight at 4 C with appropriate primary antibodies such as BiP, IRE1a, CHOP, PARP, Calnexin (Cell Signaling, Danvers, MA, USA) or Bcl-xL and Bax (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a-tubulin (AbFrontier, Seoul, Korea) and washed 3 times in TTBS. Then, the membranes were incubated with a secondary antibody (Life Technologies) for overnight at 4 C, and washed 3 times in TTBS. Quantification of the proteins assayed was performed using the CS analyzer 3.0 program (ATTO Corp., Tokyo, Japan).
2. Materials and methods 2.1. Cell culture and treatment
2.5. Measurement of GSH/GSSG
Chang (normal liver cell line) cells were kindly provided by Dr. EJ Park (Ajou University, Suwon, Korea) and were maintained in highglucose Dulbecco's modified Eagle's medium (DMEM, GE Health Care Science, Pittsburgh, PA, USA). HepG2 (human hepatocellular liver carcinoma cell line) cells were obtained from Korea Cell Line Bank (KCLB, Seoul, Korea) and were maintained in Minimum Essential Media (MEM, Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (Life Technologies) and 1% penicillinestreptomycin (Life Technologies) at 37 C in a 5% CO2 incubator. The SC-E, obtained from Jeju Technopark (JTP, Jeju, Korea), was dissolved in ethanol to obtain a stock solution of 10 mg/ ml. The stock solution was used for dilution to required concentrations. For this experiment, the cells were treated with two concentrations (75 and 150 mg/ml) of SC-E for 24 h. Vehicle control cells were treated with vehicle (1.05% ethanol) and positive control cells were treated with thapsigargin (TG, 0.1 mM, SigmaeAldrich, St Louis, MO, USA) for 24 h. ER stress inhibitors such as tauroursodeoxycholic acid (TUDCA) and sodium phenylbutyrate (4PBA) were purchased from SigmaeAldrich.
The GSH/GSSG-Glo™ Assay (Promega) is a luminescence-based system to measure GSH/GSSG ratio. After 24 h of SC-E, and TG treatment, GSH/GSSG ratio was measured using luminometer (Berthold, Bad Wildbad, Germany).
2.2. Cell viability assay Cell viability was measured using Cell Counting Kit (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan). Chang cells and HepG2 cells were seeded in 96-well plates at 5 103 cells/well.
2.6. Apoptotic cell analysis (Annexin-V/PI analysis) Annexin V/PI assay (Koma Biotech, Seoul, Korea) detects apoptotic cells using flow cytometry. After treatment of SC-E (75 mg/ml and 150 mg/ml) and TG (0.1 mM), fluorescence intensity in the cells was measured using FACS AriaII flow cytometer (BD, Franklin Lakes, NJ, USA). 2.7. Caspase-3, 7 activity Caspase-3, 7 activity was analyzed using Apo-ONE® Homogeneous Caspase-3, 7 (Promega). The cells were seeded on the blackwalled 96-well optical bottom plate at 5 103 cells/well and incubated for 24 h. Then, 100 ml of various concentrations of SC-E were added, followed by incubation for another 24 h. All the wells were treated with 50 ml of Apo-ONE® Homogeneous Caspase3, 7 buffer for 4 h at 37 C with protective lights. The fluorescence was measured with Multiple Plate Reader victor 3 (PerkinElmer, Waltham, MA, USA).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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2.8. Monitoring signal of Ca2þ (Fluo-4, AM staining) HepG2 cells were grown on a chamber slide (Thermo Fisher Scientific Inc., Waltham, MA, USA) to 40e50 % confluence. After the cells were incubated for 24 h with treatment, they were washed with phosphate-buffered saline (PBS) and stained with 4 mM Fluo4, AM (Life Technologies) for 1 h at 37 C. Then, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Subsequently, the cells were washed and mounted with mounting media and coverslip. The image was obtained by confocal laser scanning microscopy (LSM710, Carl Zeiss Microscopy GmbH, Oberkochen, Germany).
2.9. Transmission electron microscopy (TEM) analysis Cells were incubated with SC-E or TG for 24 h and washed with PBS. Then, they were fixed with Karnovsky's solution for 2 h at 4 C and washed 3 times for 10 min each with 0.05 M sodium cacodylate buffer. Next, the cells were fixed with a mixture of equal parts of 2% osmium tetroxide and 0.1 M cacodylate buffer for 2 h at 4 C and washed twice with distilled water. They were then stained with 0.5% uranyl acetate overnight at 4 C. The stained cells were subjected to dehydration with ethanol series (30%, 50%, 70%, 80%, 90%, and 100%). The dehydrated cells were embedded overnight in a mixture of equal parts of propylene oxide and Spurr's resin at room temperature. Then, the cells were embedded another overnight in
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Spurr's resin at 70 C. Ultrathin sections of the samples were cut and mounted on copper grids. The sections were stained with lead citrate and uranyl acetate and observed by TEM. All reagents were purchased from Electron Microscopy Sciences (EMS, Hatfield, PA, USA). 2.10. Measurement of the cytochrome c release The cells were seeded on 8 well-chamber slide at 7 103 cells/ well and incubated for 24 h. After the cells were incubated for 24 h with treatment, the cells were stained with Mito Tracker (Life Technologies) for 30 min at 37 C. The slides were blocked for 1 h at room temperature in Tris-buffered saline Tween (TTBS) with 3% BSA and incubated overnight at 4 C with a primary antibody, using cytochrome c (Santa Cruz Biotechnology) and washed 3 times in TTBS. Then, the slides were incubated with a secondary antibody, Alexa Fluor 488 goat anti mouse IgG (Life Technologies) for 1 h at room temperature, and washed 3 times in TTBS. The image was obtained by LSM710 (Carl Zeiss Microscopy GmbH). 2.11. Statistical analysis All data have been expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed by comparing the vehicle control and treated groups using Student's t-test. P < 0.05 was considered statistically significant.
Fig. 1. Cytotoxic effects of SC-E on the Chang and HepG2 cell lines. Chang (A, B) and HepG2 (C, D) cells were treated with diverse concentrations of SC-E (18.8e300 mg/ml for 24 h). Cell viability was measured by the CCK-8 assay. The reduction of ATP production by SC-E (18.8e300 mg/ml for 24 h) was detected in Chang (B) and HepG2 cells (D). Data are expressed as mean ± standard error of the mean (SEM). *Significantly different from control (p < 0.05), **Significantly different from control (p < 0.01), ***Significantly different from control (p < 0.001).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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Fig. 2. Generation of intracellular ROS and apoptotic cells in response to SC-E. Generation of intracellular ROS was detected by GSH/GSSG ratio (A) and apoptosis was measured by Annexin V/PI (BeD). Apoptotic cells were detected by FACS analysis (B). % of early apoptotic cells were Annexin-V positive and PI negative (C). % of apoptotic cells were Annexin-V positive, PI negative and Annexin-V positive, PI positive (D). Data are expressed as mean ± SEM. *Significantly different from vehicle control (p < 0.05), **Significantly different from Q7 vehicle control (p < 0.01), ***Significantly different from vehicle control (p < 0.001). Compared with vehicle control (*), control (#), SC-E 75 mg/ml ($) and thapsigargin (&).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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3. Results
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E treatment, more severe cytotoxicity was observed in HepG2 cells compared to Chang cells (Fig. 1A and C).
3.1. Cytotoxicity in Chang cells and HepG2 cells 3.2. ATP production in Chang cells and HepG2 cells MTT assay was performed to investigate the effect of SC-E on cell viability in Chang cells and HepG2 cells following incubation with various concentrations (18.8e300 mg/ml) of SC-E for 24 h. After SC-
ATP production was assessed after SC-E treatment to examine the effect of SC-E on mitochondrial function in HepG2 and Chang
Fig. 3. Effects of SC-E on ER stress-associated proteins and apoptosis-associated proteins in HepG2 cells. ER stress-and apoptosis-related proteins were analyzed by Western blot assay. Cells were treated with SC-E (75 and 150 mg/ml) or TG (0.1 mM) for 24 h. Each protein was analyzed using specific antibodies (A and D). The average band thickness was calculated (B and E). Caspase-3, 7 activity was increased by SC-E treatment in a concentration-dependent manner (C). Data are expressed as mean ± SEM. a-tubulin was used as a protein loading control and for band density normalization. *Significantly different from vehicle control (p < 0.05), **Significantly different from vehicle control (p < 0.01), ***Significantly different from vehicle control (p < 0.001). Compared with vehicle control (*), control (#), SC-E 75 mg/ml ($) and thapsigargin (&).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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Fig. 3. (continued).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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cells. ATP production was decreased concentration-dependently in both Chang cells and HepG2 cells (Fig. 1B and D). SC-E decreased ATP production in HepG2 cells more significantly compared with Chang cells. These results indicated that SC-E affected more severely on cell viability and ATP production in HepG2 cells than those in Chang cells. Taken together with cytotoxicity results, HepG2 cells were chosen for current study and SC-E concentration was set at 75 mg/ml and 150 mg/ml because these concentrations showed meaningful cytotoxicity in HepG2 cells. 3.3. SC-E induced oxidative stress in HepG2 cells As explained, luminescence-based GSH/GSSG-Glo™ assay was performed in order to evaluate the cellular redox status of HepG2 cells after SC-E treatment. The ratio was decreased as a function of SC-E concentration and TG (Fig. 2A). Since the ROS level and GSH/ GSSG ratio reflect the oxidative stress in cells (Lin et al., 2014) in conjunction of finding that GSH/GSSG ratio is decreased under oxidative stress (Zitka et al., 2012), SC-E seemed to affect HepG2 cell viability through oxidative stress. 3.4. SC-E induced apoptosis in HepG2 cells To determine the rate of apoptotic cell death in HepG2 cells, Annexin V/PI assay was performed after treatment with SC-E and TG (Fig. 2B). Early-stage apoptotic cells were Annexin V positive, and PI negative. Late-stage apoptotic cells were Annexin V positive, and PI positive. The rate of apoptotic cell death in the cells was increased significantly by SC-E and TG (Fig. 2C and D). 3.5. SC-E induced apoptosis through ER stress Western blot assay was performed to determine the protein level of ER stress and apoptosis. We found that the protein expression level of ER stress and apoptosis was increased in HepG2 cells after SC-E treatment in a concentration-dependent manner (Fig. 3A and D). These results were reconfirmed by densitometric
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analysis (Fig. 3B and E). SC-E increased ER stress-related proteins expression. SC-E also increased Bax protein expression and PARP cleavage concentration-dependently. However, anti-apoptotic protein Bcl-xL was decreased (Fig. 3D and E). Caspase-3, 7 activity was increased by SC-E treatment in a concentration-dependent manner (Fig. 3C). Release of cytochrome c from mitochondria induced mitochondria-mediated apoptosis, thus, known to cause early cell death (Boris et al., 1998). SC-E induced the release of cy- Q1 tochrome c from mitochondria to cytosol (Fig. S1). Taken altogether, our data indicate that SC-E induces mitochondria-mediated apoptosis in conjunction with ER stress. 3.6. Suppression of endoplasmic reticulum stress reduced apoptosis induced by SC-E Western blot assay was performed to determine whether SC-E affected ER stress-related proteins or apoptosis-related proteins. When the cells were pretreated with 4-PBA, TUDCA as ER stress inhibitors, apoptosis induced by SC-E was reduced significantly (Fig. 4A and B). These ER stress-mediated effects on apoptosis were reconfirmed by caspase-3, 7 activity assay (Fig. 4C). In addition, increase of intracellular free calcium level induced by SC-E was significantly reduced by 4-PBA and TUDCA (Fig. 5). These results support that SC-E induces apoptosis through ER stress. 3.7. SC-E elevated intracellular free Ca2þ Intracellular calcium level is increased by the release of Ca2þ from the ER to the cytosol in the circumstances of cell damage or ER stress. To measure intracellular Ca2þ level, HepG2 cells was exposed to SC-E (75 and 150 mg/ml), TG (0.1 mM) and co-treatment with 4PBA, TUDCA. Then, the cells were stained with Fluo-4, AM. SC-E increased Fluo-4, AM fluorescence (Green) in a concentrationdependent manner compared to untreated control cells. However, co-treatment 4-PBA and TUDCA decreased Fluo-4, AM fluorescence (Fig. 5).
Fig. 4. Effects of ER stress inhibitor, 4-PBA and TUDCA on levels of ER stress-related proteins and apoptosis-related proteins in SC-E treated cells. ER stress-related proteins and apoptosis-related proteins were analyzed by Western blot assay. Cells were treated with SC-E (150 mg/ml) or TG (0.1 mM) and co-treatment ER stress inhibitors: 4-PBA (10 mM) and TUDCA (5 mM). Each protein was analyzed using specific antibodies (A and B). Caspase-3, 7 activity was decreased by co-treatment with 4-PBA and TUDCA (C). Data are expressed as mean ± SEM. *Significantly different from SC-E 150 mg/ml (p < 0.05), **Significantly different from thapsigargin (p < 0.01).
Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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Fig. 5. Microscopic fluorescence imaging of Fluo-4, AM staining for intracellular Ca2þ level after SC-E treatment. HepG2 cells were treated SC-E (75 and 150 mg/ml), TG (0.1 mM) and co-treatment ER stress inhibitors, 4-PBA (10 mM) and TUDCA (5 mM), and then cells stained with Fluo-4, AM (Green). Scale bar ¼ 10 mm.
3.8. SC-E damaged ER and mitochondrial morphology Extracellular stimuli cause organelle morphology changes that lead to cell dysfunction. TEM analysis was performed to examine whether SC-E treatment caused morphological changes in HepG2
cells. The TEM image distinctly showed morphological changes, such as ER dilation and mitochondrial crista damage at 75 mg/ml SC-E and critical ER swelling and mitochondrial crista and outer membrane damage at 150 mg/ml SC-E, compared to untreated control cells (Fig. 6).
Fig. 6. TEM analysis of the cell response to SC-E. Transmission electron microscopy of unexposed (control) cells (A), and of cells at 24 h after treatment with SC-E 75 mg/ml (B), SC-E 150 mg/ml (C) and TG 0.1 mM (D).N; nuclear, M; mitochondria, ER; endoplasmic reticulum, G; Golgi apparatus. Scale marker indicates 2 mm and 0.5 mm.
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4. Discussion Recently, cancer cell-specific cell death has been proposed as a new targeted treatment for anticancer therapy (Liu et al., 2014). There is a long history of anticancer drugs identified from herbal medicines, and naturally derived compounds can be a source of powerful anticancer agents. Some studies have reported the inhibition of melanoma formation and cell death in prostate and breast cancer cells using S. chinensis Baill constituents. The compound manassantin A isolated from SC-E has anti-proliferative effect in different cancer cell lines (Rao and Reddy, 1990; Feng et al., 2011; Lee et al., 2012). However, there were no studies on ER stressmediated apoptosis regarding SC-E (Kim et al., 2011; Lee et al., 2013). This is why current study has focused on the function of SC-E as a potential effective anticancer reagent through ER stressmediated apoptosis. Many lines of evidences have shown that ROS production is an important factor for apoptosis induction. ROS, which is continually generated and eliminated in biological systems, plays a role in diverse biological functions in the normal state but also induces pathological progression in abnormal state (Yang et al., 2014). Imbalance between generation and elimination leads to overproduction of ROS, which could induce apoptosis by cell death signaling (Li et al., 2015). Other studies have mentioned the role of ROS in caspase activation and ER stress during apoptosis (Pierre et al., 2013; Tagawa et al., 2008). Our current results also showed the evidence that oxidative stress was associated with ER stress in SC-E treated cells. ER stress is well known as a crucial part of the regulation of cellular homeostasis, especially in the unfolded protein response (UPR) (Chen et al., 2014). Binding immunoglobulin protein (BiP), also called GRP78, is a glucose-regulated protein and is one of the most efficient ER luminal chaperone proteins. BiP is associated with three ER transmembrane proteins, IRE1a, PERK, and ATF6, during unstressed conditions (Dara et al., 2011; Wang et al., 2014). Under ER stress, BiP is activated, UPR signal is initiated, and the three ER transmembrane proteins are dissociated from BiP. Activated IRE1a produces spliced XBP1 by splitting XBP fragments and activates proapoptotic proteins through activation of the signaling cascade controlling cell survival or death. CHOP is a member of C/EBP family and known to be induced highly under ER stress (Fig. 3A and B). The activation of IRE1a, PERK and ATF6 has been reported to increase CHOP protein expression, activating and initiating the ER stressinduced apoptotic cascade (Qu et al., 2013; Chen et al., 2014; Liu et al., 2014; Yang et al., 2014). We observed that the protein expression levels of IRE1a and CHOP were increased after treatment with SC-E in HepG2 cells. Our data suggested that SC-E treatment induced ER stress and apoptosis. Our results also demonstrated that the increase of cytosolic Ca2þ concentration was associated with ER stress (Fig. 5). In fact, recent works have suggested that ER calcium depletion by TG probably triggers apoptosis while overexpression of the anti-apoptotic Bcl-2 protein, which anchors the intracellular membranes, maintains the Ca2þ homeostasis in ER, thereby preventing apoptosis induction by TG (Yoshida et al., 2006). Moreover, the release of Ca2þ from the ER has known to play an important relation between the ER and the major mechanism in controlling apoptosis (Li et al., 2012a,b; Cao et al., 2013). Then, the influx of Ca2þ to the mitochondria activates the release of cytochrome c that causes apoptosis. All things taken together, our results showed that SC-E mediated the increase of cytosolic Ca2þ concentration through ER stress. Caspases have been reported to play an essential role in apoptosis or scheduled cell death. The release of cytochrome c to the cytosol in apoptosis along the mitochondrial pathway is related to activation of downstream caspases, especially caspase-3, which
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are crucial in DNA fragmentation associated with morphological changes and apoptosis of mitochondria (Kim et al., 2009; Li et al., 2012a,b). Furthermore, caspase-3 is necessary for PARP cleavage, an important marker of apoptosis. Both cytochrome c-related apoptosis and cytochrome c release are mainly controlled by members of the Bcl-2 family. Bax and Bcl-2 are recognized as important regulators. Bax inserts into the mitochondrial outer membrane and translocates to the mitochondria to facilitate the release of cytochrome c (Huang et al., 2006). Although the association of Bcl-2 and Bax neutralizes the proapoptotic effects, a large increase in Bax expression induces apoptosis. Our results demonstrated that SC-E was an inducer of apoptosis in HepG2 cells. SC-Einduced apoptosis was dependent on the activation of mitochondria-mediated caspases and on the interaction between Bax and the mitochondria (Fig. 3D and E). In conclusion, ER stress-mediated apoptosis induced by SC-E could inhibit the survival of hepatocellular carcinoma HepG2 cells. Our results demonstrated that SC-E induced ER stress in HepG2 cells, disrupted intracellular calcium homeostasis, and led to cell death through apoptosis by mitochondrial damage. We propose that SC-E could be used as an anticancer agent for liver cancer. Acknowledgments This work was supported by the Bio-Synergy Research Project (NRF-2012M3A9C4048819) of the Ministry of Science, ICT and Future Planning through the National Research Foundation. CLSM images were acquired at National Center for Inter-University Research Facilities in Seoul National University. This study was supported through BK21 PLUS Program for Creative Veterinary Science Research. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fct.2015.05.008. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2015.05.008. Uncited references Zhivotovsky et al., 1998. References Cao, A., Li, Q., Yin, P., Dong, Y., Shi, H., Wang, L., Ji, G., Xie, J., Wu, D., 2013. Curcumin induces apoptosis in human gastric carcinoma AGS cells and colon carcinoma HT-29 cells through mitochondrial dysfunction and endoplasmic reticulum stress. Apoptosis 18 (11), 1391e1402. Chang, J.S., Lee, S.W., Kim, M.S., Yun, B.R., Park, M.H., Lee, S.G., Park, S.J., Lee, W.S., Rho, M.C., 2011. Manassantin A and B from Saururus chinensis inhibit interleukin-6-induced signal transducer and activator of transcription 3 activation in Hep3B cells. J. Pharmacol. Sci. 115 (1), 84e88. Chen, Y., Gui, D., Chen, J., He, D., Luo, Y., Wang, N., 2014. Down-regulation of PERKATF4-CHOP pathway by Astragaloside IV is associated with the inhibition of endoplasmic reticulum stress-induced podocyte apoptosis in diabetic rats. Cell. Physiol. Biochem. 33 (6), 1975e1987. Dara, L., Ji, C., Kaplowitz, N., 2011. The contribution of endoplasmic reticulum stress to liver diseases. Hepatology 53 (5), 1752e1763. Feng, Y., Wang, N., Zhu, M., Feng, Y., Li, H., Tsao, S., 2011. Recent progress on anticancer candidates in patents of herbal medicinal products. Recent Pat. Food Nutr. Agric. 3 (1), 30e48. Heath-Engel, H.M., Chang, N.C., Shore, G.C., 2008. The endoplasmic reticulum in apoptosis and autophagy: role of the BCL-2 protein family. Oncogene 27 (50), 6419e6433. Huang, Y.T., Huang, D.M., Chueh, S.C., Teng, C.M., Guh, J.H., 2006. Alisol B acetate, a triterpene from Alismatis rhizoma, induces Bax nuclear translocation and
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Please cite this article in press as: Lee, A.Y., et al., Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food and Chemical Toxicology (2015), http://dx.doi.org/10.1016/j.fct.2015.05.008
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