- Email: [email protected]
Compound K induced apoptosis via endoplasmic reticulum Ca2+ release through ryanodine receptor in human lung cancer cells
Accepted Manuscript Compound K induced apoptosis via endoplasmic reticulum Ca ryanodine receptor in human lung cancer cells
2+
release through
Dong-Hyun Shin, Dong-Gyu Leem, Ji-Sun Shin, Joo-Il Kim, Kyung-Tack Kim, Sang Yoon Choi, Myung-Hee Lee, Jung-Hye Choi, Kyung-Tae Lee, PhD. PII:
S1226-8453(16)30252-4
DOI:
10.1016/j.jgr.2017.01.015
Reference:
JGR 254
To appear in:
Journal of Ginseng Research
Received Date: 29 October 2016 Accepted Date: 31 January 2017
Please cite this article as: Shin D-H, Leem D-G, Shin J-S, Kim J-I, Kim K-T, Choi SY, Lee M-H, Choi 2+ J-H, Lee K-T, Compound K induced apoptosis via endoplasmic reticulum Ca release through ryanodine receptor in human lung cancer cells, Journal of Ginseng Research (2017), doi: 10.1016/ j.jgr.2017.01.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Compound K induced apoptosis via endoplasmic reticulum Ca2+ release
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through ryanodine receptor in human lung cancer cells
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Dong-Hyun Shin1, Dong-Gyu Leem1,3, Ji-Sun Shin1, Joo-Il Kim1, Kyung-Tack Kim2,
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Sang Yoon Choi2, Myung-Hee Lee2, Jung-Hye Choi3,4, Kyung-Tae Lee1,3*
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Seoul 130-701, Republic of Korea 2
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Republic of Korea 3
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Traditional Food Research Center, Korea Food Research Institute, Seongnam 13539,
Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
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Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
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Corresponding author: Kyung-Tae Lee, PhD.
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Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University,
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Department of Pharmaceutical Biochemistry, College of Pharmacy
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Kyung Hee University
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Dongdaemun-Ku, Hoegi-Dong, Seoul, 130-701,
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Republic of Korea
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Tel.: +82 2 961 0860; Fax: +82 2 966 3885.
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E-mail : [email protected]
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Running title: Compound K induces apoptosis via ER Ca2+ release
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ACCEPTED MANUSCRIPT Abstract
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Background: Extended endoplasmic reticulum (ER) stress may initiate apoptotic pathways in
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cancer cells, and ER stress has been reported to possibly increase tumor death in cancer
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therapy. We previously reported that caspase-8 played an important role in compound K-
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induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage,
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cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The
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mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the
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role of endoplasmic reticulum (ER) stress have not yet been understood.
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Methods: The apoptotic effects of compound K were analyzed using flow cytometry, and the
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changes in protein levels were determined using a Western blot analysis. The intracellular
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calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM.
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Results: Compound K-induced ER stress was confirmed through increased phosphorylation
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of eIF2α and protein levels of GRP78/BiP, XBP-1S, and IRE1α in human lung cancer cells.
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Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-
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calpain activities that were both significantly inhibited by pretreatment either with BAPTA-
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AM (an intracellular Ca2+ chelator) or dantrolene (a RyR channel antagonist). These results
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were correlated with the outcome that compound K induced ER stress-related apoptosis
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through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12) partially ameliorated
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this effect. Interestingly, 4-PBA (ER stress inhibitor) dramatically improved the compound K-
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induced apoptosis.
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Conclusion: Cell survival and intracellular Ca2+ homeostasis during ER stress in human lung
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cancer cells are important factors in the induction of the compound K-induced apoptotic
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pathway.
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Keywords: Apoptosis, Calcium, Compound K, ER stress, Lung cancer cells 2
ACCEPTED MANUSCRIPT 1. Introduction
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Apoptosis plays a pivotal role in the process of physiological cell deletion to maintain the
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balance between cellular replication and death. Some agents possessing chemotherapeutics
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and chemoprevention properties have exhibited their ability by inducing either a cell cycle
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arrest or apoptotic cell death. Therefore, the induction of apoptosis is an attractive strategy for
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treatment of cancer [1, 2]. Apoptotic signaling can be initiated through two pathways: (i) the
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extrinsic pathway via death receptors, such as, tumor necrosis factor receptor 1 (TNFR1) and
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FAS, which is activated by ligation of certain ligands. Activation of death receptor results in
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initiation of the caspase-8-dependent cell death process [3]; and (ii) an intrinsic pathway via
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mitochondria. Intrinsic apoptosis pathway is mediated by the release of cytochrome c from
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mitochondria, which triggers the activation of caspase-9, cleaves caspase-3, and ultimately
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promotes apoptotic cell death [4]. Therefore, activation of caspases is a critical event in the
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progressions of apoptosis.
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Endoplasmic reticulum (ER) is not only a reservoir for intracellular Ca2+, but also a home
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for folding and posttranslational modifications of secretory proteins [5, 6]. ER stress occurs
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when ER is subjected to adverse situations such as hypoxia, nutrient deprivation, fails of
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posttranslational modifications, imbalance in Ca2+ homeostasis, or an increased accumulation
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of unfolded proteins [7]. ER stress causes the unfolded protein response (UPR) to enhance the
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protein folding capacity and decrease protein synthesis, and UPR activation from ER stress
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initiates intracellular signaling pathways for cell protection.
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The ER stress response is mainly regulated by three ER transmembrane proteins: activating
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transcription factor-6 (ATF-6), inositol requiring kinase-1 (IRE-1), and protein kinase-like
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endoplasmic reticulum kinase (PERK) [8]. Under usual conditions, these transmembrane
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proteins keep inactive bound with ER-resident chaperone glucose-regulated protein-78
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(GRP78/BiP). The UPR is regulated by an ER stress sensor IRE1α [9]. Activation of IRE1α, a 3
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protein-1 (XBP-1) mRNA. This process encodes a transcriptional activator, which initiates
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the transcription of chaperone protein-encoding genes, whose products have a function of ER
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protein folding [10]. PERK, another ER stress transducer, is a transmembrane kinase that
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phosphorylates eukaryotic translation initiation factor 2 subunit α (eIF2α), resulting in
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reduction of protein synthesis and counteraction of ER protein overload [11]. Active
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fragments of ATF-6 translocate into the nucleus and bind to the ER stress response element
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thereby stimulating transcription of transcription factor genes (e.g. XBP-1) and ER chaperone
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genes (e.g. GRP78) as well as expression of CCAAT/enhancer-binding protein-homologous
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protein (CHOP/GADD153) [7]. Severe or sustained ER stress can provokes pro-apoptotic
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ATF-6, PERK, and IRE-1 signaling and induces CHOP increase. Furthermore, caspase-12
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participates in ER stress-mediated apoptosis, as shown by the resistance to ER stress-induced
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apoptosis in caspase-12 knockdown mice [12].
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The intracellular Ca2+ level handles a various cellular phenomenon including transcription,
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exocytosis, apoptosis, and proliferation [13], and its concentration is under the control of ion
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channels, multiple pumps, and binding proteins [14]. The Ca2+ release from the ER is
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mediated through the ryanodine receptors (RyRs) and ER-resident inositol trisphosphate
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receptors (IP3Rs) [15]. However, even with tight regulation of Ca2+ release from the ER, the
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exhaustion of ER Ca2+ and the overload of cytosolic Ca2+ can occur due to several stimuli.
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The disrupted Ca2+ homeostasis and unchecked increases in cytosolic Ca2+ can induce
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apoptosis via the activation of ER resident caspases and the activation of processes in the
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cytoplasm [16-18].
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Ginseng, the root and rhizome of Panax ginseng Meyer, has been widely adapted in traditional medicine in East Asia. Ginsenosides are major bioactive components in ginseng and describe a various group of steroidal saponins. More than 20 ginsenosides have been 4
ACCEPTED MANUSCRIPT reported to possess a variety of biological properties including neuroprotective, anticancer
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and anti-inflammatory activities [19]. The two major sub-types of ginsenosides have been
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termed protopanaxadiols and protopanaxatriols, which after ingestion can give rise to novel
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metabolites in the body [16, 17]. 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol
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(compound K) (Fig. 1A) is a major metabolite of several protopanaxadiol-type ginsenosides,
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including Rb1, Rb2 and Rc, by intestinal bacteria through the multistage cleavage of sugar
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moieties [20]. It has been investigated in terms of its anti-diabetic [21-23], anti-inflammatory
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[24-26] and anticancer [27, 28] effects.
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Although a previous study suggested a role for ER stress in the apoptosis in colon cancer cells
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[29], the role of Ca2+ released from ER and caspase-12 in compound K-induced apoptosis in
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human lung cancer cells has not yet been reported. We report that compound K induces Ca2+
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release from ER via the RyR channel, leading to the activation of m-calpain, caspase-12 and
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caspase-3. In addition, there is an increase in the levels of the endoplasmic reticular stress
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markers, IRE1α, XBP-1S and GRP78.
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2. Materials and methods
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2.1. Chemicals and reagents Compound K (purity >98%) used in present study was isolated from P. ginseng, and its
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structural identities were determined spectroscopically (1H and 13NMR, IR, MS) as
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previously described (25). RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS),
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penicillin, streptomycin and Fura-2/AM were obtained from Life Technologies Inc. 3-(4,5-
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Dimethylthiazol-2-yl)- 2,5-diphenyl-tertazolium bromide (MTT), dimethyl sulfoxide
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(DMSO), RNase A, phenylmethylsulfonylfluoride (PMSF), propidium iodide (PI), 4-
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phenylbutyrate (4-PBA), 2-Aminoethoxydiphenyl Borate (2-APB), Dantrolene, BAPTA-AM,
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ethylene glycol tetraacetic acid (EGTA) and CGP37157 were purchased from Sigma
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Chemical Co. The following antibodies for caspase-3, caspase-7, caspase-9, poly (ADP-
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ribose) polymerase (PARP), XBP-1, c-FLIPL, Bid, Bcl-2, Bcl-xL, p-eIF2α, GADD 153, m-
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calpain and β-actin were purchased from Santa Cruz Biotechnology, Inc. The antibodies for
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X-linked inhibitor of apoptosis protein (XIAP), caspase-8 and caspase-12 were purchased
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from BD Biosciences, Pharmingen. The antibody for IRE1α and GRP78 were purchased
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from Cell Signaling Technology. z-VAD-fmk, z-ATAD-fmk were purchased from
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Calbiochem.
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2.2. Cell culture and sample treatment Human lung adenocarcinoma A549 and human lung squamous cell carcinoma SK-MES-
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1 were purchased from the Korea Cell Line Bank (Seoul, Republic of Korea). A549 was
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grown in RPMI-1640 medium with 10% FBS, penicillin (100 units/ml) and streptomycin
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sulfate (100 µg/ml) in a humidified atmosphere (5% CO2, 95% humidity, 37°C). SK-MES-1
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was grown in DMEM medium with 10% FBS, penicillin (100 units/ml) and streptomycin 6
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sulfate (100 µg/ml) in a humidified atmosphere (5% CO2, 95% humidity, 37°C). Cells were
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incubated for 48 h with various concentrations (5, 10, 15 µM) or with 15 µM of compound K
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for various times (6, 12, 24, 36, or 48 h) in 2% FBS contained medium.
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2.3. Determination of cytotoxicity
The cytotoxicity was assessed through an MTT assay. Briefly, the cells (0.5 × 105 cells/ml) were plated for 24 h in each well containing 100 µl of the RPMI or DMEM medium at 96-
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well plates. Then, cells were treated with various concentrations of compound K and
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incubated for 48 h. After 48 h, 20 µl of MTT [5 mg/ml stock solution in phosphate-buffered
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saline (PBS)] were added and further incubated for 4 h. The medium was removed and
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formazan blue formed in the cells was dissolved with dimethyl sulfoxide (200 µl). The
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optical density was measured at 540 nm.
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2.4. Quantification of apoptosis via PI and Annexin V double staining
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Cells treated with Compound K were collected and were suspended with 100 µl of binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Cell staining were
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conducted with 5 µl of PI (50 µg/ml) and 5 µl of FITC-conjugated Annexin V for 30 min at
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room temperature in dark place. Thereafter, 400 µl of binding buffer were added for analysis
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via fluorescence-activated cell sorting (FACS) cater-plus flow cytometry (Becton Dickinson
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Co, Heidelberg, Germany).
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2.5. Preparation of cellular protein and Western blot analysis Cells treated with Compound K were washed twice with ice-cold PBS and were
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centrifuged at 200 g for 5 min. The obtained cell pellet was suspended in PRO-PREPTM
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protein extraction buffer (Intron, Seoul, Republic of Korea). Equal amounts of protiens were 7
ACCEPTED MANUSCRIPT separated via SDS-PAGE and were transferred to PVDF membranes for Western blot
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analysis. The immunoblot was incubated specific primary antibodies overnight at 4°C. HRP-
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conjugated secondary antibodies were detected using an enhanced chemiluminescence
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detection system (GE healthcare, WI, USA).
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2.6. Calcium quantification
A549 and SK-MES-1 cells grown on cover glass were incubated overnight. Cytosolic free
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Ca2+ was measured using the Ca2+-sensitive fluorescent indicator dye Fura-2/AM. Cells
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grown on a matrigel-coated cover-slide bottom dish were washed three times with PBS and
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were incubated in the dark for 30 min at room temperature with Fura-2/AM (final
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concentration 1 µM) in PBS. The cells were washed again with PBS three times and were
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analyzed by being illuminated with an alternating light of 340 and 380 nm from a rotating
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filter wheel.
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2.7. Detection of calcium concentration
Free cytosolic calcium was measured by a fluorescence Ca2+ indicator Fluo-3/AM. Treated
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cells were washed twice with HBSS (130 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 10 mM
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HEPES, 10 mM glucose, 2 mM CaCl2, pH 7.4), re-suspended, and then incubated with the
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Fluo-3/AM (3 mM) for 30 min. The free cytosolic Ca2+ levels were analyzed by flow
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cytometry (Becton Dickinson Co, Heidelberg, Germany).
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2.8. Statistical analysis
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The results are expressed as the mean ± S.D. of triplicate experiments. Statistically
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significant values were compared using ANOVA and Dunnett’s post hoc test. Statistical
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analysis was performed using SigmaPlot software version 10.0 (Systat Software, Inc., San
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Jose, CA, USA). P-values of less than 0.05 were considered to be statistically significant.
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3. Results
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3.1. Compound K induced caspase-dependent apoptosis in human lung cancer cells.
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We examined the effect of compound K on the cell viabilities using MTT assays in human lung cancer cells. Compound K was shown to have cytotoxicity on human lung
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adenocarcinoma A549 and squamous lung carcinoma SK-MES-1 cells (IC50: 17.78 µM and
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16.53 µM, respectively). To further investigate whether the cytotoxic effect of compound K
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was associated with the induction of apoptosis, we estimated the translocation of
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phosphatidylserine (PS) using Annexin V and PI double staining. The percentage of Annexin
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V-positive cells was found to increase in a time- and concentration-dependent manner after
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treatment with compound K in A549 and SK-MES-1 cells (Fig. 1B).
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To establish the mechanism associated with compound K-induced apoptosis, we examined the activation of caspase-8, -9, -3 and cleavage of PARP (an endogenous substrate of
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caspase-3) in A549 and SK-MES-1 cells. Compound K increased procaspase-8, -9, -3 and
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PARP-1 cleavage in a time- and concentration-dependent manner (Fig. 1C). To investigate
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the involvement of caspases in compound K-induced apoptosis, A549 and SK-MES-1 cells
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were pretreated with z-VAD-fmk (a broad caspase inhibitor) for 1 h and were then treated
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with compound K for 48 h. As shown in Fig. 1D and 1E, z-VAD-fmk significantly
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suppressed compound K-induced apoptosis in A549 and SK-MES-1 cells. These
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observations indicate that compound K-induced apoptosis involved the caspase-dependent
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pathway in A549 and SK-MES-1 cells. As expected, the expressions of anti-apoptotic
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proteins c-FLIPL, X-linked IAP (XIAP), Bcl-2, and Bcl-xL were substantially decreased with
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compound K treatment in both cell lines in a time- and concentration-dependent manner (Fig.
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1E), indicating concomitant activation of both the extrinsic and the intrinsic pathways of
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apoptosis.
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3.2. Compound K induced ER stress and led to activation of the unfolded protein response
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(UPR).
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Because ER stress is deeply involved in the induction of apoptosis [22], we assess the effect of the compound K on ER stress markers. IRE1α activation has been shown to promote
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the splicing of XBP1 mRNA in a 26 nucleotide intron and bring about its spliced variant
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XBP-1S [30]. A previous study indicated XBP1 activation was one of main characteristics of
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ER stress [9]. Treatment of A549 and SK-MES-1 cells for various times with compound K
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(5-15 µM) sustainedly increased the levels of IRE1α and XBP-1S in both cells (Fig. 2). Next,
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we determined the compound K-induced changes in other two streams of ER stress signaling
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(ATF-6/p-eIF2α and PERK/CHOP). Western blots of compound K-treated cells revealed a
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significant increase of p-eIF2α in A549 and SK-MES-1 cells. However, an increase in CHOP
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protein was not observed in either of the cells (Fig. 2). The upregulation of molecular
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chaperones further confirmed in compound K-induced ER stress; this included increased
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GRP78 level, which is sensitive to alterations in the redox state of ER (Fig. 2).
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3.3. Compound K induced apoptosis through caspase-12-dependent pathway.
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Caspase-12 is located in the ER and responsible for ER stress-induced apoptosis [12]. Caspase-12 is cleaved and activated specifically during ER stress, but not by death receptor-
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or mitochondrial-mediated apoptotic signals. Subsequently, activated caspase-12 may induce
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the activation of executor caspases. This has prompted us to assess whether ER stress is
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induced after compound K treatment by evaluating the levels of caspase-12 cleavage by
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Western blotting in A549 and SK-MES-1 human lung cancer cells. We found that both a
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time- and concentration-dependent increase in caspase-12 cleavage (Fig. 3A). In addition, the
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treatment of z-ATAD-fmk (a specific caspase-12 inhibitor) restrain the compound K-induced
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cleavage of caspase-12 and subsequent activation of caspase-3 (Fig. 3B), confirming that
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compound K-induced apoptosis was caspase-12-dependent in those cells. Caspases-12 is unique to ER and caspase-3 is a link executor caspase for ER stress and
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apoptosis pathways. Thus, our result implied a crosstalk between ER stress and the extrinsic
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and/or intrinsic pathways of apoptosis, with caspase-12 being the upstream signaling
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molecule to caspase-3. Further, as shown in Fig. 3C, z-ATAD-fmk partially inhibited
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compound K-induced apoptosis, which implied that compound K induced apoptosis in
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human lung cancer cells via both caspase-12 and extrinsic and/or intrinsic signaling pathways
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[12].
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3.4. Compound K increased the intracellular calcium levels and led to m-calpain activation. To investigate whether the compound K-induced apoptosis involved changes in intracellular Ca2+ levels ([Ca2+]i), we measured the change in intensity of the intracellular
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Ca2+-sensitive dye Fura-2/AM in A549 and SK-MES-1 cells before and after treatment.
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Treatment with compound K resulted in a significant increase in [Ca2+]i in A549 and SK-
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MES-1 cells (Fig. 4A).
Since caspase-12 activation has been reported to be possibly mediated by calcium-
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dependent cysteine protease, m-calpain, in response to calcium release from the ER during
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ER stress [31], we examined whether compound K influenced the m-calpain activation by
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Western blotting. Fig. 3A shows that compound K promoted the cleavage of m-calpain to its
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active fragments in a time- and concentration-dependent manner. With the ER being the most
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important intracellular Ca2+ store [5], regulation of [Ca2+]i by the ER is mainly mediated by
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Ca2+ uptake into the ER through SERCA Ca2+ pumps and Ca2+ release through Ca2+ channels,
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such as IP3Rs or RyRs [12, 32, 33]. To determine whether compound K-induced [Ca2+]i in
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A549 and SK-MES-1 cells were caused by ER Ca2+-related channels, RyRs and IP3Rs were
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blocked by Ca2+ channel blockers dantrolene and 2-aminoethoxydiphenyl borate (2-APB),
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confirmed using BAPTA-AM (intracellular Ca2+ chelator). As shown in Figs. 4B-4D,
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dantrolene or BAPTA-AM treatment markedly attenuated compound K-induced apoptosis,
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whereas 2-APB did not. Next, we measured the m-calpain, caspase-12, and caspase-3
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activation of cells treated with compound K in presence or absence of BAPTA-AM or
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dantrolene to reveal the role of increased [Ca2+]i in compound K-induced ER stress. As
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shown Figs. 4E-4G, the chelation of cytosolic Ca2+ by treatment with BAPTA-AM or
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dantrolene suppressed the compound K-induced cleavage of m-calpain, caspase-12, and
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caspase-3, whereas 2-APB did not activate caspase-3. Taken together, these data suggested
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that RyR-mediated Ca2+ release is involved in compound K-induced ER stress and apoptotic
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cell death.
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3.5. Inhibition of ER stress enhanced compound K-induced apoptosis. Next, we examined the connection between compound K-induced ER stress and apoptosis
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in human lung cancer A549 and SK-MES-1 cells. We used 4-PBA (ER stress inhibitor) to
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alleviate ER stress in these lung cancer cells treated with compound K. Pretreatment with 4-
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PBA reduced the expression of compound K-induced ER-specific proteins, such as GRP78,
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IRE1α, and p-eIF2α (Fig. 5A). We then estimated the changes of apoptosis-associated
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proteins including caspase-12, m-calpain, and caspase-3. The results indicate that compound
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K-induced activation of caspase-12, m-calpain and caspase-3 were clearly upregulated by 4-
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PBA (Figs. 5A). Importantly, inhibition of ER stress by 4-PBA significantly enhanced the
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apoptosis in compound K-treated A549 and SK-MES-1 cells at 24 h (Fig. 5B). In a manner
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consistent with the induction of apoptosis, inhibition of ER stress significantly increased
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compound K-induced [Ca2+]i levels by 4-PBA (Fig. 5C). These results demonstrated that
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inhibition of ER stress enhanced apoptosis via increase of [Ca2+]i levels in A549 and SK-
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MES-1 cells treated with compound K. Taken together, these results indicate that induction of
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ER stress participated in protecting the cell against compound K-induced apoptosis.
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4. Discussion Apoptosis is an essential cellular response to eliminate the genetically mutated and hyper-
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proliferative cells, thereby reducing the cancer risk. Therefore, defects in systemic regulation
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of apoptosis could cause carcinogenesis and chemoresistance [28]. As mentioned above,
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apoptosis can induce through two main pathways, the “extrinsic” pathway mediated by death
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receptor and the “intrinsic” pathway mediated by mitochondria, and truncated Bid protein
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serves as a link between “intrinsic” and “extrinsic” pathway [34]. Caspases supervise both
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pathways either directly or indirectly to induce the cleavages of cellular proteins, a feature of
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apoptosis [35]. Our previous study has shown that caspase-8 contributes to compound K-
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induced apoptosis by activating caspase-3 and -9 and modulating Bcl-2 families in HL-60
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human leukemia cells [25].
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In the present study, we confirmed that compound K induced caspase-dependent apoptosis in A549 and SK-MES-1 human lung cancer cells. Since the Bcl-2 family proteins play a
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significant role in apoptosis, we studied the effect of compound K on the expression of Bcl-2
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family proteins in A549 and SK-MES-1. Compound K was found to down-regulated the
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levels of anti-apoptotic proteins, including c-FLIPL, XIAP, Bcl-2 and Bcl-xL, leading to
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apoptotic pathways via both intrinsic and extrinsic activation. Our present results confirm
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those of Lee et al. [21], in as much as compound K showed cytotoxic and caspase-dependent
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apoptosis-inducing activities in human lung cancer cells.
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ER is a central organelle responsible for cytosolic Ca2+ signals as an intracellular calcium
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store and for optimal protein processing, such as assembling and folding [33]. Loss of
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balance between ER protein folding load and capacity in different physiological and
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pathological conditions bring about accumulation of unfolded proteins in the ER lumen,
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known as an ER stress condition. ER stress-mediated UPR modulates the expression of
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IRE1α, PERK, GRP78/BiP, and ATF-6/p-eIF2α. Mild to moderate ER stress boosts cell 15
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survival through UPR to reduce ER stress [36], whereas severe and prolonged ER stress or
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inhibition of ER stress causes cell death in some cancer types including lung cancer [35].
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Despite considerable studies on compound K-induced apoptosis in several cancer cell lines [21-23, 37], little is known about its cytotoxic mechanisms in terms of ER stress-related
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apoptosis. The this study, UPR-related proteins were induced in compound K-treated lung
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cancer cells as: (i) compound K increased IRE1α and XBP-1S protein levels; (ii) compound
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K induced increase of GRP78 expression; and (iii) compound K induced phosphorylation of
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eIF2α. However, the increase of CHOP protein, an activating protein of apoptosis in ER
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stress, did not detected. This implies that mild ER stress induced by compound K could not
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induce ER stress-associated apoptosis in human lung cancer cells.
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There is a growing body of evidence that the release of Ca2+ from ER and consequent increase in cytosolic Ca2+ levels can play pivotal roles in regulating cell survival and
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apoptosis in a variety of cell types [38]. In addition, the dysregulation of ER Ca2+
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homeostasis occurs as an early event during many forms of apoptosis and has been
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implicated in the pathophysiology of several diseases, including cancer. In the present study,
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the release of ER calcium stores resulted in an increase in cytosolic free Ca2+ levels,
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suggesting the disruption of intracellular Ca2+ homeostasis, which leads to the promotion of
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cell dysfunction and apoptosis in A549 and SK-MES-1 cells.
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The elevation of [Ca2+]i or depletion of ER Ca2+ stores are typical ER stress responses of
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cells [33]. Cytosolic Ca2+ binds and activates Ca2+-dependent intracellular cysteine protease,
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m-calpain, caspase-12 and then the apoptotic pathway [12, 39]. The experimental evidence
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presented here showed that the evaluation of [Ca2+]i by efflux from endoplasmic reticulum
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caused by compound K triggered apoptosis through the activation of m-calpain and caspase-
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12 pathway in human lung cancer cells.
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The elevated [Ca2+]i may be due to an influx of Ca2+ from the extracellular medium across 16
ACCEPTED MANUSCRIPT the plasma membrane and/or Ca2+ release from intracellular stores, mainly from the ER. We
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demonstrated the function of extracellular Ca2+ in the increase in [Ca2+]i after compound K
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treatment in A549 and SK-MES-1 cells by analyzing [Ca2+]i in cells placed in a medium
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without Ca2+. There were no significant differences in increase of cytosolic Ca2+ by presence
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or absence of Ca2+ in medium (data not shown), indicating that an increased [Ca2+]i by
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compound K does not flowed from extracellular medium in our model. Zhang et al. also
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previously demonstrated that elevated Ca2+ levels by compound K in cytosolic and
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mitochondrial may be mediated by Ca2+ release from ER stores into the cytosol of human
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colon cancer cells [40]. We further investigated whether the increase in [Ca2+]i after
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compound K treatment in A549 and SK-MES-1 cells was also resulted from the Ca2+
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release from the ER via the IP3R and/or RyR channels, which have been involved in
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apoptotic Ca2+ signaling in several models [41]. We thus treated the cells with inhibitors of
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these receptors, dantrolene and 2-APB, respectively. Pretreatment of dantrolene inhibited
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[Ca2+]i levels and attenuated compound K-induced apoptosis, whereas 2-APB did not.
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Furthermore, BAPTA-AM, a permeant Ca2+ chelator, was treated to investigate the possible
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role of [Ca2+]i in the progression of compound K-induced apoptosis. Preincubation with
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BAPTA-AM also significantly suppressed compound K-induced apoptosis through
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inactivation of m-calpain, procaspase-12, and caspase-3. Therefore, we noticed that increased
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[Ca2+]i levels through RyR channel, instead of that from IP3R channel, were closely
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associated with compound K-induced apoptosis. However, further experiments are needed to
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examine how ER stress and increased [Ca2+]i activates the mitochondrial apoptosis pathway.
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To further determine the biological function of ER stress on apoptosis in compound K-
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treated A549 and SK-MES-1 cells, we blocked ER stress in human lung cancer cells by using
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ER stress inhibitor 4-PBA. Compound K-induced apoptosis was found to have been
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obviously enhanced in A549 and SK-MES-1 cells treated with 4-PBA. In a situation of 17
ACCEPTED MANUSCRIPT enhanced m-calpain and caspase-12 activation by 4-PBA, compound K-induced UPR may
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participate in intracellular Ca2+ modulation. Based on these results, we found that UPR was
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related to cell protection. Also, these results could support the premise that UPR signals
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induced by compound K were involved in a cell protective effect against the observed
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apoptosis [42].
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In summary, it was found that compound K could induce ER stress and apoptosis, and ER stress was observed to be involved in compound K-induced protection in A549 and SK-
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MES-1 human lung cancer cells. Exposure to compound K could also be concluded to cause
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cell death by triggering ER Ca2+ release via RyR. Thus, the obligatory signaling molecules
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for ER stress, UPR, and calcium release might be considered to be the molecular targets for
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compound K-induced apoptosis.
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Acknowledgements
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This work was supported by the research grant from the Korea Food Research Institute
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(E0145202).
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Disclosures The authors have no conflicts of interest.
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Fig. 1. Compound K induced apoptosis in human lung cancer cells. (A) The chemical
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structure of compound K. (B) Cells were treated with various concentrations (5, 10, 15 µM)
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of compound K for the indicated times. Cells were co-stained with PI and FITC-conjugated
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annexin V, and the translocation of phosphatidylserine (PS) was detected by flow cytometry.
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(C) Compound K induced apoptosis via caspase activation and downregulation of anti-
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apoptotic proteins in A549 and SK-MES-1 cells. β-Actin was used as an internal control. (D)
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The effect of the broad-spectrum caspase inhibitor (z-VAD-fmk) on apoptosis was
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determined by co-staining with PI and FITC-conjugated annexin V, and the translocation of
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phosphatidylserine (PS) was detected by flow cytometry. (E) c-FLIPL, XIAP, Bcl-2 and Bcl-
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xL were analyzed by Western blotting and β-actin was used as an internal control. Data are
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presented as means ± SD of three independent experiments. **P < 0.01, ***P < 0.001 vs. the
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compound K treated group.
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Fig. 2. Compound K induced ER-stress and activation of unfolded protein response. Cells
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were treated with various concentrations (5, 10, 15 µM) of compound K (48 h) and various
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times (0, 6, 12, 24, 36, 48 h) with compound K (15 µM). GRP78, IRE1α, XBP-1S, p-eIF2α
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and CHOP were analyzed by Western blotting and β-actin was used as an internal control.
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Fig. 3. Compound K induced apoptosis via caspase-12 activation. (A) Cells were treated with
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various concentrations (5, 10, 15 µM) of compound K (48 h) and various times (0, 6, 12, 24,
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36, 48 h) with compound K (15 µM). Caspase-12 and m-calpain were analyzed by Western
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blotting and β-actin was used as an internal control. (B) Cells were pretreated with 1 µM z-
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ATAD-fmk for 1 h and then treated with 15 µM of compound K for 48 h. Caspase-12 and
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procaspase-3 were analyzed by Western blotting and β-actin was used as an internal control. 25
ACCEPTED MANUSCRIPT (C) The effect of the caspase-12 inhibitor (z-ATAD-fmk) on apoptosis was determined by co-
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staining with PI and FITC-conjugated annexin V, and the translocation of phosphatidylserine
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(PS) was detected by flow cytometry. Data are presented as means ± SD of three independent
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experiments. ***P < 0.001 vs. the compound K treated group.
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Fig. 4. Compound K induced apoptosis via Ca2+ mediated caspase-12 activation. (A)
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Cytosolic Ca2+ levels were measured by Fura-2/AM fluorescence dye. (B, C, D) Effect of the
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RyR channel antagonist (Dantrolene), IP3R channel antagonist (2-APB) or Ca2+ chelator
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(BAPTA-AM) on apoptosis was determined by testing the co-staining with PI and FITC-
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conjugated annexin V, and the translocation of phosphatidylserine (PS) was detected by flow
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cytometry. Data are presented as means ± SD of three independent experiments. ***P <
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0.001 vs. the compound K treated group. (E, F) Cells were pretreated with 5 µM dantrolene
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or 2.5 µM BAPTA-AM for 1 h and then treated with 15 µM of compound K for 48 h. m-
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Calpain, caspase-12 and procaspase-3 were analyzed by Western blotting and β-actin was
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used as an internal control. (G) Cells were pretreated with 30 µM 2-APB for 1 h and were
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then treated with 15 µM of compound K for 48 h. Procaspase-3 were analyzed by Western
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blotting and β-actin was used as an internal control.
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Fig. 5. Effects of 4-PBA on compound K induced ER stress, [Ca2+]i and apoptosis. (A) Cells
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were pretreated with 4 mM 4-PBA for 1 h and were then treated with 15 µM of compound K
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for 48 h. GRP78, IRE1α, p-eIF2α, m-calpain, caspase-12, and procaspase-3 were analyzed by
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Western blotting and β-actin was used as an internal control. (B) Effect of the 4-PBA on
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apoptosis was determined by testing the co-staining with PI and FITC-conjugated annexin V,
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and the translocation of phosphatidylserine (PS) was detected by flow cytometry. (C)
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Cytosolic Ca2+ levels were measured by fluo-3 AM fluorescence dye and were measured 26
ACCEPTED MANUSCRIPT using a flow cytometry. 4-PBA was pretreated for 1 h and then treated compound K. Data are
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presented as means ± SD of three independent experiments. ***P < 0.001 vs. the compound
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K-treated group.
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2+
release through
Dong-Hyun Shin, Dong-Gyu Leem, Ji-Sun Shin, Joo-Il Kim, Kyung-Tack Kim, Sang Yoon Choi, Myung-Hee Lee, Jung-Hye Choi, Kyung-Tae Lee, PhD. PII:
S1226-8453(16)30252-4
DOI:
10.1016/j.jgr.2017.01.015
Reference:
JGR 254
To appear in:
Journal of Ginseng Research
Received Date: 29 October 2016 Accepted Date: 31 January 2017
Please cite this article as: Shin D-H, Leem D-G, Shin J-S, Kim J-I, Kim K-T, Choi SY, Lee M-H, Choi 2+ J-H, Lee K-T, Compound K induced apoptosis via endoplasmic reticulum Ca release through ryanodine receptor in human lung cancer cells, Journal of Ginseng Research (2017), doi: 10.1016/ j.jgr.2017.01.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Compound K induced apoptosis via endoplasmic reticulum Ca2+ release
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through ryanodine receptor in human lung cancer cells
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Dong-Hyun Shin1, Dong-Gyu Leem1,3, Ji-Sun Shin1, Joo-Il Kim1, Kyung-Tack Kim2,
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Sang Yoon Choi2, Myung-Hee Lee2, Jung-Hye Choi3,4, Kyung-Tae Lee1,3*
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Seoul 130-701, Republic of Korea 2
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Republic of Korea 3
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Traditional Food Research Center, Korea Food Research Institute, Seongnam 13539,
Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
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Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
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Corresponding author: Kyung-Tae Lee, PhD.
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Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University,
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Department of Pharmaceutical Biochemistry, College of Pharmacy
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Kyung Hee University
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Dongdaemun-Ku, Hoegi-Dong, Seoul, 130-701,
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Republic of Korea
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Tel.: +82 2 961 0860; Fax: +82 2 966 3885.
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E-mail : [email protected]
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Running title: Compound K induces apoptosis via ER Ca2+ release
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ACCEPTED MANUSCRIPT Abstract
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Background: Extended endoplasmic reticulum (ER) stress may initiate apoptotic pathways in
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cancer cells, and ER stress has been reported to possibly increase tumor death in cancer
29
therapy. We previously reported that caspase-8 played an important role in compound K-
30
induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage,
31
cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The
32
mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the
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role of endoplasmic reticulum (ER) stress have not yet been understood.
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Methods: The apoptotic effects of compound K were analyzed using flow cytometry, and the
35
changes in protein levels were determined using a Western blot analysis. The intracellular
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calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM.
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Results: Compound K-induced ER stress was confirmed through increased phosphorylation
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of eIF2α and protein levels of GRP78/BiP, XBP-1S, and IRE1α in human lung cancer cells.
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Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-
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calpain activities that were both significantly inhibited by pretreatment either with BAPTA-
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AM (an intracellular Ca2+ chelator) or dantrolene (a RyR channel antagonist). These results
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were correlated with the outcome that compound K induced ER stress-related apoptosis
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through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12) partially ameliorated
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this effect. Interestingly, 4-PBA (ER stress inhibitor) dramatically improved the compound K-
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induced apoptosis.
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Conclusion: Cell survival and intracellular Ca2+ homeostasis during ER stress in human lung
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cancer cells are important factors in the induction of the compound K-induced apoptotic
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pathway.
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Keywords: Apoptosis, Calcium, Compound K, ER stress, Lung cancer cells 2
ACCEPTED MANUSCRIPT 1. Introduction
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Apoptosis plays a pivotal role in the process of physiological cell deletion to maintain the
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balance between cellular replication and death. Some agents possessing chemotherapeutics
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and chemoprevention properties have exhibited their ability by inducing either a cell cycle
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arrest or apoptotic cell death. Therefore, the induction of apoptosis is an attractive strategy for
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treatment of cancer [1, 2]. Apoptotic signaling can be initiated through two pathways: (i) the
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extrinsic pathway via death receptors, such as, tumor necrosis factor receptor 1 (TNFR1) and
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FAS, which is activated by ligation of certain ligands. Activation of death receptor results in
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initiation of the caspase-8-dependent cell death process [3]; and (ii) an intrinsic pathway via
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mitochondria. Intrinsic apoptosis pathway is mediated by the release of cytochrome c from
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mitochondria, which triggers the activation of caspase-9, cleaves caspase-3, and ultimately
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promotes apoptotic cell death [4]. Therefore, activation of caspases is a critical event in the
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progressions of apoptosis.
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Endoplasmic reticulum (ER) is not only a reservoir for intracellular Ca2+, but also a home
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for folding and posttranslational modifications of secretory proteins [5, 6]. ER stress occurs
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when ER is subjected to adverse situations such as hypoxia, nutrient deprivation, fails of
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posttranslational modifications, imbalance in Ca2+ homeostasis, or an increased accumulation
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of unfolded proteins [7]. ER stress causes the unfolded protein response (UPR) to enhance the
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protein folding capacity and decrease protein synthesis, and UPR activation from ER stress
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initiates intracellular signaling pathways for cell protection.
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The ER stress response is mainly regulated by three ER transmembrane proteins: activating
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transcription factor-6 (ATF-6), inositol requiring kinase-1 (IRE-1), and protein kinase-like
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endoplasmic reticulum kinase (PERK) [8]. Under usual conditions, these transmembrane
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proteins keep inactive bound with ER-resident chaperone glucose-regulated protein-78
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(GRP78/BiP). The UPR is regulated by an ER stress sensor IRE1α [9]. Activation of IRE1α, a 3
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protein-1 (XBP-1) mRNA. This process encodes a transcriptional activator, which initiates
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the transcription of chaperone protein-encoding genes, whose products have a function of ER
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protein folding [10]. PERK, another ER stress transducer, is a transmembrane kinase that
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phosphorylates eukaryotic translation initiation factor 2 subunit α (eIF2α), resulting in
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reduction of protein synthesis and counteraction of ER protein overload [11]. Active
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fragments of ATF-6 translocate into the nucleus and bind to the ER stress response element
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thereby stimulating transcription of transcription factor genes (e.g. XBP-1) and ER chaperone
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genes (e.g. GRP78) as well as expression of CCAAT/enhancer-binding protein-homologous
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protein (CHOP/GADD153) [7]. Severe or sustained ER stress can provokes pro-apoptotic
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ATF-6, PERK, and IRE-1 signaling and induces CHOP increase. Furthermore, caspase-12
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participates in ER stress-mediated apoptosis, as shown by the resistance to ER stress-induced
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apoptosis in caspase-12 knockdown mice [12].
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exocytosis, apoptosis, and proliferation [13], and its concentration is under the control of ion
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channels, multiple pumps, and binding proteins [14]. The Ca2+ release from the ER is
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mediated through the ryanodine receptors (RyRs) and ER-resident inositol trisphosphate
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receptors (IP3Rs) [15]. However, even with tight regulation of Ca2+ release from the ER, the
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exhaustion of ER Ca2+ and the overload of cytosolic Ca2+ can occur due to several stimuli.
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The disrupted Ca2+ homeostasis and unchecked increases in cytosolic Ca2+ can induce
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apoptosis via the activation of ER resident caspases and the activation of processes in the
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cytoplasm [16-18].
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Ginseng, the root and rhizome of Panax ginseng Meyer, has been widely adapted in traditional medicine in East Asia. Ginsenosides are major bioactive components in ginseng and describe a various group of steroidal saponins. More than 20 ginsenosides have been 4
ACCEPTED MANUSCRIPT reported to possess a variety of biological properties including neuroprotective, anticancer
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and anti-inflammatory activities [19]. The two major sub-types of ginsenosides have been
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termed protopanaxadiols and protopanaxatriols, which after ingestion can give rise to novel
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metabolites in the body [16, 17]. 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol
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(compound K) (Fig. 1A) is a major metabolite of several protopanaxadiol-type ginsenosides,
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including Rb1, Rb2 and Rc, by intestinal bacteria through the multistage cleavage of sugar
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moieties [20]. It has been investigated in terms of its anti-diabetic [21-23], anti-inflammatory
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[24-26] and anticancer [27, 28] effects.
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Although a previous study suggested a role for ER stress in the apoptosis in colon cancer cells
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[29], the role of Ca2+ released from ER and caspase-12 in compound K-induced apoptosis in
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human lung cancer cells has not yet been reported. We report that compound K induces Ca2+
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release from ER via the RyR channel, leading to the activation of m-calpain, caspase-12 and
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caspase-3. In addition, there is an increase in the levels of the endoplasmic reticular stress
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markers, IRE1α, XBP-1S and GRP78.
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2. Materials and methods
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2.1. Chemicals and reagents Compound K (purity >98%) used in present study was isolated from P. ginseng, and its
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structural identities were determined spectroscopically (1H and 13NMR, IR, MS) as
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previously described (25). RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS),
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penicillin, streptomycin and Fura-2/AM were obtained from Life Technologies Inc. 3-(4,5-
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Dimethylthiazol-2-yl)- 2,5-diphenyl-tertazolium bromide (MTT), dimethyl sulfoxide
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(DMSO), RNase A, phenylmethylsulfonylfluoride (PMSF), propidium iodide (PI), 4-
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phenylbutyrate (4-PBA), 2-Aminoethoxydiphenyl Borate (2-APB), Dantrolene, BAPTA-AM,
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ethylene glycol tetraacetic acid (EGTA) and CGP37157 were purchased from Sigma
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Chemical Co. The following antibodies for caspase-3, caspase-7, caspase-9, poly (ADP-
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ribose) polymerase (PARP), XBP-1, c-FLIPL, Bid, Bcl-2, Bcl-xL, p-eIF2α, GADD 153, m-
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calpain and β-actin were purchased from Santa Cruz Biotechnology, Inc. The antibodies for
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X-linked inhibitor of apoptosis protein (XIAP), caspase-8 and caspase-12 were purchased
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from BD Biosciences, Pharmingen. The antibody for IRE1α and GRP78 were purchased
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from Cell Signaling Technology. z-VAD-fmk, z-ATAD-fmk were purchased from
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Calbiochem.
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2.2. Cell culture and sample treatment Human lung adenocarcinoma A549 and human lung squamous cell carcinoma SK-MES-
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1 were purchased from the Korea Cell Line Bank (Seoul, Republic of Korea). A549 was
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grown in RPMI-1640 medium with 10% FBS, penicillin (100 units/ml) and streptomycin
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sulfate (100 µg/ml) in a humidified atmosphere (5% CO2, 95% humidity, 37°C). SK-MES-1
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was grown in DMEM medium with 10% FBS, penicillin (100 units/ml) and streptomycin 6
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sulfate (100 µg/ml) in a humidified atmosphere (5% CO2, 95% humidity, 37°C). Cells were
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incubated for 48 h with various concentrations (5, 10, 15 µM) or with 15 µM of compound K
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for various times (6, 12, 24, 36, or 48 h) in 2% FBS contained medium.
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2.3. Determination of cytotoxicity
The cytotoxicity was assessed through an MTT assay. Briefly, the cells (0.5 × 105 cells/ml) were plated for 24 h in each well containing 100 µl of the RPMI or DMEM medium at 96-
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well plates. Then, cells were treated with various concentrations of compound K and
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incubated for 48 h. After 48 h, 20 µl of MTT [5 mg/ml stock solution in phosphate-buffered
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saline (PBS)] were added and further incubated for 4 h. The medium was removed and
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formazan blue formed in the cells was dissolved with dimethyl sulfoxide (200 µl). The
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optical density was measured at 540 nm.
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2.4. Quantification of apoptosis via PI and Annexin V double staining
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Cells treated with Compound K were collected and were suspended with 100 µl of binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Cell staining were
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conducted with 5 µl of PI (50 µg/ml) and 5 µl of FITC-conjugated Annexin V for 30 min at
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room temperature in dark place. Thereafter, 400 µl of binding buffer were added for analysis
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via fluorescence-activated cell sorting (FACS) cater-plus flow cytometry (Becton Dickinson
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Co, Heidelberg, Germany).
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2.5. Preparation of cellular protein and Western blot analysis Cells treated with Compound K were washed twice with ice-cold PBS and were
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centrifuged at 200 g for 5 min. The obtained cell pellet was suspended in PRO-PREPTM
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protein extraction buffer (Intron, Seoul, Republic of Korea). Equal amounts of protiens were 7
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analysis. The immunoblot was incubated specific primary antibodies overnight at 4°C. HRP-
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conjugated secondary antibodies were detected using an enhanced chemiluminescence
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detection system (GE healthcare, WI, USA).
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2.6. Calcium quantification
A549 and SK-MES-1 cells grown on cover glass were incubated overnight. Cytosolic free
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Ca2+ was measured using the Ca2+-sensitive fluorescent indicator dye Fura-2/AM. Cells
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grown on a matrigel-coated cover-slide bottom dish were washed three times with PBS and
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were incubated in the dark for 30 min at room temperature with Fura-2/AM (final
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concentration 1 µM) in PBS. The cells were washed again with PBS three times and were
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analyzed by being illuminated with an alternating light of 340 and 380 nm from a rotating
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filter wheel.
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2.7. Detection of calcium concentration
Free cytosolic calcium was measured by a fluorescence Ca2+ indicator Fluo-3/AM. Treated
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cells were washed twice with HBSS (130 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 10 mM
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HEPES, 10 mM glucose, 2 mM CaCl2, pH 7.4), re-suspended, and then incubated with the
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Fluo-3/AM (3 mM) for 30 min. The free cytosolic Ca2+ levels were analyzed by flow
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cytometry (Becton Dickinson Co, Heidelberg, Germany).
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2.8. Statistical analysis
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The results are expressed as the mean ± S.D. of triplicate experiments. Statistically
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significant values were compared using ANOVA and Dunnett’s post hoc test. Statistical
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analysis was performed using SigmaPlot software version 10.0 (Systat Software, Inc., San
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Jose, CA, USA). P-values of less than 0.05 were considered to be statistically significant.
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3. Results
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3.1. Compound K induced caspase-dependent apoptosis in human lung cancer cells.
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We examined the effect of compound K on the cell viabilities using MTT assays in human lung cancer cells. Compound K was shown to have cytotoxicity on human lung
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adenocarcinoma A549 and squamous lung carcinoma SK-MES-1 cells (IC50: 17.78 µM and
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16.53 µM, respectively). To further investigate whether the cytotoxic effect of compound K
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was associated with the induction of apoptosis, we estimated the translocation of
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phosphatidylserine (PS) using Annexin V and PI double staining. The percentage of Annexin
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V-positive cells was found to increase in a time- and concentration-dependent manner after
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treatment with compound K in A549 and SK-MES-1 cells (Fig. 1B).
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To establish the mechanism associated with compound K-induced apoptosis, we examined the activation of caspase-8, -9, -3 and cleavage of PARP (an endogenous substrate of
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caspase-3) in A549 and SK-MES-1 cells. Compound K increased procaspase-8, -9, -3 and
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PARP-1 cleavage in a time- and concentration-dependent manner (Fig. 1C). To investigate
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the involvement of caspases in compound K-induced apoptosis, A549 and SK-MES-1 cells
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were pretreated with z-VAD-fmk (a broad caspase inhibitor) for 1 h and were then treated
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with compound K for 48 h. As shown in Fig. 1D and 1E, z-VAD-fmk significantly
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suppressed compound K-induced apoptosis in A549 and SK-MES-1 cells. These
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observations indicate that compound K-induced apoptosis involved the caspase-dependent
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pathway in A549 and SK-MES-1 cells. As expected, the expressions of anti-apoptotic
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proteins c-FLIPL, X-linked IAP (XIAP), Bcl-2, and Bcl-xL were substantially decreased with
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compound K treatment in both cell lines in a time- and concentration-dependent manner (Fig.
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1E), indicating concomitant activation of both the extrinsic and the intrinsic pathways of
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apoptosis.
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3.2. Compound K induced ER stress and led to activation of the unfolded protein response
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(UPR).
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Because ER stress is deeply involved in the induction of apoptosis [22], we assess the effect of the compound K on ER stress markers. IRE1α activation has been shown to promote
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the splicing of XBP1 mRNA in a 26 nucleotide intron and bring about its spliced variant
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XBP-1S [30]. A previous study indicated XBP1 activation was one of main characteristics of
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ER stress [9]. Treatment of A549 and SK-MES-1 cells for various times with compound K
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(5-15 µM) sustainedly increased the levels of IRE1α and XBP-1S in both cells (Fig. 2). Next,
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we determined the compound K-induced changes in other two streams of ER stress signaling
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(ATF-6/p-eIF2α and PERK/CHOP). Western blots of compound K-treated cells revealed a
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significant increase of p-eIF2α in A549 and SK-MES-1 cells. However, an increase in CHOP
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protein was not observed in either of the cells (Fig. 2). The upregulation of molecular
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chaperones further confirmed in compound K-induced ER stress; this included increased
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GRP78 level, which is sensitive to alterations in the redox state of ER (Fig. 2).
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3.3. Compound K induced apoptosis through caspase-12-dependent pathway.
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Caspase-12 is located in the ER and responsible for ER stress-induced apoptosis [12]. Caspase-12 is cleaved and activated specifically during ER stress, but not by death receptor-
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or mitochondrial-mediated apoptotic signals. Subsequently, activated caspase-12 may induce
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the activation of executor caspases. This has prompted us to assess whether ER stress is
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induced after compound K treatment by evaluating the levels of caspase-12 cleavage by
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Western blotting in A549 and SK-MES-1 human lung cancer cells. We found that both a
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time- and concentration-dependent increase in caspase-12 cleavage (Fig. 3A). In addition, the
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treatment of z-ATAD-fmk (a specific caspase-12 inhibitor) restrain the compound K-induced
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cleavage of caspase-12 and subsequent activation of caspase-3 (Fig. 3B), confirming that
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compound K-induced apoptosis was caspase-12-dependent in those cells. Caspases-12 is unique to ER and caspase-3 is a link executor caspase for ER stress and
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apoptosis pathways. Thus, our result implied a crosstalk between ER stress and the extrinsic
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and/or intrinsic pathways of apoptosis, with caspase-12 being the upstream signaling
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molecule to caspase-3. Further, as shown in Fig. 3C, z-ATAD-fmk partially inhibited
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compound K-induced apoptosis, which implied that compound K induced apoptosis in
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human lung cancer cells via both caspase-12 and extrinsic and/or intrinsic signaling pathways
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[12].
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3.4. Compound K increased the intracellular calcium levels and led to m-calpain activation. To investigate whether the compound K-induced apoptosis involved changes in intracellular Ca2+ levels ([Ca2+]i), we measured the change in intensity of the intracellular
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Ca2+-sensitive dye Fura-2/AM in A549 and SK-MES-1 cells before and after treatment.
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Treatment with compound K resulted in a significant increase in [Ca2+]i in A549 and SK-
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MES-1 cells (Fig. 4A).
Since caspase-12 activation has been reported to be possibly mediated by calcium-
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dependent cysteine protease, m-calpain, in response to calcium release from the ER during
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ER stress [31], we examined whether compound K influenced the m-calpain activation by
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Western blotting. Fig. 3A shows that compound K promoted the cleavage of m-calpain to its
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active fragments in a time- and concentration-dependent manner. With the ER being the most
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important intracellular Ca2+ store [5], regulation of [Ca2+]i by the ER is mainly mediated by
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Ca2+ uptake into the ER through SERCA Ca2+ pumps and Ca2+ release through Ca2+ channels,
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such as IP3Rs or RyRs [12, 32, 33]. To determine whether compound K-induced [Ca2+]i in
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A549 and SK-MES-1 cells were caused by ER Ca2+-related channels, RyRs and IP3Rs were
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blocked by Ca2+ channel blockers dantrolene and 2-aminoethoxydiphenyl borate (2-APB),
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confirmed using BAPTA-AM (intracellular Ca2+ chelator). As shown in Figs. 4B-4D,
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dantrolene or BAPTA-AM treatment markedly attenuated compound K-induced apoptosis,
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whereas 2-APB did not. Next, we measured the m-calpain, caspase-12, and caspase-3
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activation of cells treated with compound K in presence or absence of BAPTA-AM or
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dantrolene to reveal the role of increased [Ca2+]i in compound K-induced ER stress. As
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shown Figs. 4E-4G, the chelation of cytosolic Ca2+ by treatment with BAPTA-AM or
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dantrolene suppressed the compound K-induced cleavage of m-calpain, caspase-12, and
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caspase-3, whereas 2-APB did not activate caspase-3. Taken together, these data suggested
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that RyR-mediated Ca2+ release is involved in compound K-induced ER stress and apoptotic
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cell death.
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3.5. Inhibition of ER stress enhanced compound K-induced apoptosis. Next, we examined the connection between compound K-induced ER stress and apoptosis
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in human lung cancer A549 and SK-MES-1 cells. We used 4-PBA (ER stress inhibitor) to
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alleviate ER stress in these lung cancer cells treated with compound K. Pretreatment with 4-
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PBA reduced the expression of compound K-induced ER-specific proteins, such as GRP78,
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IRE1α, and p-eIF2α (Fig. 5A). We then estimated the changes of apoptosis-associated
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proteins including caspase-12, m-calpain, and caspase-3. The results indicate that compound
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K-induced activation of caspase-12, m-calpain and caspase-3 were clearly upregulated by 4-
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PBA (Figs. 5A). Importantly, inhibition of ER stress by 4-PBA significantly enhanced the
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apoptosis in compound K-treated A549 and SK-MES-1 cells at 24 h (Fig. 5B). In a manner
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consistent with the induction of apoptosis, inhibition of ER stress significantly increased
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compound K-induced [Ca2+]i levels by 4-PBA (Fig. 5C). These results demonstrated that
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inhibition of ER stress enhanced apoptosis via increase of [Ca2+]i levels in A549 and SK-
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MES-1 cells treated with compound K. Taken together, these results indicate that induction of
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ER stress participated in protecting the cell against compound K-induced apoptosis.
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4. Discussion Apoptosis is an essential cellular response to eliminate the genetically mutated and hyper-
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proliferative cells, thereby reducing the cancer risk. Therefore, defects in systemic regulation
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of apoptosis could cause carcinogenesis and chemoresistance [28]. As mentioned above,
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apoptosis can induce through two main pathways, the “extrinsic” pathway mediated by death
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receptor and the “intrinsic” pathway mediated by mitochondria, and truncated Bid protein
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serves as a link between “intrinsic” and “extrinsic” pathway [34]. Caspases supervise both
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pathways either directly or indirectly to induce the cleavages of cellular proteins, a feature of
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apoptosis [35]. Our previous study has shown that caspase-8 contributes to compound K-
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induced apoptosis by activating caspase-3 and -9 and modulating Bcl-2 families in HL-60
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human leukemia cells [25].
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In the present study, we confirmed that compound K induced caspase-dependent apoptosis in A549 and SK-MES-1 human lung cancer cells. Since the Bcl-2 family proteins play a
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significant role in apoptosis, we studied the effect of compound K on the expression of Bcl-2
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family proteins in A549 and SK-MES-1. Compound K was found to down-regulated the
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levels of anti-apoptotic proteins, including c-FLIPL, XIAP, Bcl-2 and Bcl-xL, leading to
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apoptotic pathways via both intrinsic and extrinsic activation. Our present results confirm
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those of Lee et al. [21], in as much as compound K showed cytotoxic and caspase-dependent
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apoptosis-inducing activities in human lung cancer cells.
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ER is a central organelle responsible for cytosolic Ca2+ signals as an intracellular calcium
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store and for optimal protein processing, such as assembling and folding [33]. Loss of
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balance between ER protein folding load and capacity in different physiological and
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pathological conditions bring about accumulation of unfolded proteins in the ER lumen,
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known as an ER stress condition. ER stress-mediated UPR modulates the expression of
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IRE1α, PERK, GRP78/BiP, and ATF-6/p-eIF2α. Mild to moderate ER stress boosts cell 15
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survival through UPR to reduce ER stress [36], whereas severe and prolonged ER stress or
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inhibition of ER stress causes cell death in some cancer types including lung cancer [35].
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Despite considerable studies on compound K-induced apoptosis in several cancer cell lines [21-23, 37], little is known about its cytotoxic mechanisms in terms of ER stress-related
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apoptosis. The this study, UPR-related proteins were induced in compound K-treated lung
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cancer cells as: (i) compound K increased IRE1α and XBP-1S protein levels; (ii) compound
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K induced increase of GRP78 expression; and (iii) compound K induced phosphorylation of
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eIF2α. However, the increase of CHOP protein, an activating protein of apoptosis in ER
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stress, did not detected. This implies that mild ER stress induced by compound K could not
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induce ER stress-associated apoptosis in human lung cancer cells.
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There is a growing body of evidence that the release of Ca2+ from ER and consequent increase in cytosolic Ca2+ levels can play pivotal roles in regulating cell survival and
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apoptosis in a variety of cell types [38]. In addition, the dysregulation of ER Ca2+
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homeostasis occurs as an early event during many forms of apoptosis and has been
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implicated in the pathophysiology of several diseases, including cancer. In the present study,
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the release of ER calcium stores resulted in an increase in cytosolic free Ca2+ levels,
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suggesting the disruption of intracellular Ca2+ homeostasis, which leads to the promotion of
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cell dysfunction and apoptosis in A549 and SK-MES-1 cells.
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The elevation of [Ca2+]i or depletion of ER Ca2+ stores are typical ER stress responses of
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cells [33]. Cytosolic Ca2+ binds and activates Ca2+-dependent intracellular cysteine protease,
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m-calpain, caspase-12 and then the apoptotic pathway [12, 39]. The experimental evidence
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presented here showed that the evaluation of [Ca2+]i by efflux from endoplasmic reticulum
343
caused by compound K triggered apoptosis through the activation of m-calpain and caspase-
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12 pathway in human lung cancer cells.
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The elevated [Ca2+]i may be due to an influx of Ca2+ from the extracellular medium across 16
ACCEPTED MANUSCRIPT the plasma membrane and/or Ca2+ release from intracellular stores, mainly from the ER. We
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demonstrated the function of extracellular Ca2+ in the increase in [Ca2+]i after compound K
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treatment in A549 and SK-MES-1 cells by analyzing [Ca2+]i in cells placed in a medium
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without Ca2+. There were no significant differences in increase of cytosolic Ca2+ by presence
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or absence of Ca2+ in medium (data not shown), indicating that an increased [Ca2+]i by
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compound K does not flowed from extracellular medium in our model. Zhang et al. also
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previously demonstrated that elevated Ca2+ levels by compound K in cytosolic and
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mitochondrial may be mediated by Ca2+ release from ER stores into the cytosol of human
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colon cancer cells [40]. We further investigated whether the increase in [Ca2+]i after
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compound K treatment in A549 and SK-MES-1 cells was also resulted from the Ca2+
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release from the ER via the IP3R and/or RyR channels, which have been involved in
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apoptotic Ca2+ signaling in several models [41]. We thus treated the cells with inhibitors of
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these receptors, dantrolene and 2-APB, respectively. Pretreatment of dantrolene inhibited
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[Ca2+]i levels and attenuated compound K-induced apoptosis, whereas 2-APB did not.
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Furthermore, BAPTA-AM, a permeant Ca2+ chelator, was treated to investigate the possible
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role of [Ca2+]i in the progression of compound K-induced apoptosis. Preincubation with
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BAPTA-AM also significantly suppressed compound K-induced apoptosis through
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inactivation of m-calpain, procaspase-12, and caspase-3. Therefore, we noticed that increased
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[Ca2+]i levels through RyR channel, instead of that from IP3R channel, were closely
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associated with compound K-induced apoptosis. However, further experiments are needed to
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examine how ER stress and increased [Ca2+]i activates the mitochondrial apoptosis pathway.
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To further determine the biological function of ER stress on apoptosis in compound K-
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treated A549 and SK-MES-1 cells, we blocked ER stress in human lung cancer cells by using
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ER stress inhibitor 4-PBA. Compound K-induced apoptosis was found to have been
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obviously enhanced in A549 and SK-MES-1 cells treated with 4-PBA. In a situation of 17
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participate in intracellular Ca2+ modulation. Based on these results, we found that UPR was
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related to cell protection. Also, these results could support the premise that UPR signals
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induced by compound K were involved in a cell protective effect against the observed
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apoptosis [42].
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In summary, it was found that compound K could induce ER stress and apoptosis, and ER stress was observed to be involved in compound K-induced protection in A549 and SK-
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MES-1 human lung cancer cells. Exposure to compound K could also be concluded to cause
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cell death by triggering ER Ca2+ release via RyR. Thus, the obligatory signaling molecules
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for ER stress, UPR, and calcium release might be considered to be the molecular targets for
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compound K-induced apoptosis.
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Acknowledgements
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This work was supported by the research grant from the Korea Food Research Institute
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(E0145202).
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Disclosures The authors have no conflicts of interest.
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Fig. 1. Compound K induced apoptosis in human lung cancer cells. (A) The chemical
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structure of compound K. (B) Cells were treated with various concentrations (5, 10, 15 µM)
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of compound K for the indicated times. Cells were co-stained with PI and FITC-conjugated
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annexin V, and the translocation of phosphatidylserine (PS) was detected by flow cytometry.
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(C) Compound K induced apoptosis via caspase activation and downregulation of anti-
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apoptotic proteins in A549 and SK-MES-1 cells. β-Actin was used as an internal control. (D)
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The effect of the broad-spectrum caspase inhibitor (z-VAD-fmk) on apoptosis was
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determined by co-staining with PI and FITC-conjugated annexin V, and the translocation of
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phosphatidylserine (PS) was detected by flow cytometry. (E) c-FLIPL, XIAP, Bcl-2 and Bcl-
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xL were analyzed by Western blotting and β-actin was used as an internal control. Data are
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presented as means ± SD of three independent experiments. **P < 0.01, ***P < 0.001 vs. the
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compound K treated group.
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Fig. 2. Compound K induced ER-stress and activation of unfolded protein response. Cells
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were treated with various concentrations (5, 10, 15 µM) of compound K (48 h) and various
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times (0, 6, 12, 24, 36, 48 h) with compound K (15 µM). GRP78, IRE1α, XBP-1S, p-eIF2α
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and CHOP were analyzed by Western blotting and β-actin was used as an internal control.
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Fig. 3. Compound K induced apoptosis via caspase-12 activation. (A) Cells were treated with
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various concentrations (5, 10, 15 µM) of compound K (48 h) and various times (0, 6, 12, 24,
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36, 48 h) with compound K (15 µM). Caspase-12 and m-calpain were analyzed by Western
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blotting and β-actin was used as an internal control. (B) Cells were pretreated with 1 µM z-
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ATAD-fmk for 1 h and then treated with 15 µM of compound K for 48 h. Caspase-12 and
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procaspase-3 were analyzed by Western blotting and β-actin was used as an internal control. 25
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staining with PI and FITC-conjugated annexin V, and the translocation of phosphatidylserine
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(PS) was detected by flow cytometry. Data are presented as means ± SD of three independent
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experiments. ***P < 0.001 vs. the compound K treated group.
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Fig. 4. Compound K induced apoptosis via Ca2+ mediated caspase-12 activation. (A)
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Cytosolic Ca2+ levels were measured by Fura-2/AM fluorescence dye. (B, C, D) Effect of the
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RyR channel antagonist (Dantrolene), IP3R channel antagonist (2-APB) or Ca2+ chelator
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(BAPTA-AM) on apoptosis was determined by testing the co-staining with PI and FITC-
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conjugated annexin V, and the translocation of phosphatidylserine (PS) was detected by flow
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cytometry. Data are presented as means ± SD of three independent experiments. ***P <
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0.001 vs. the compound K treated group. (E, F) Cells were pretreated with 5 µM dantrolene
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or 2.5 µM BAPTA-AM for 1 h and then treated with 15 µM of compound K for 48 h. m-
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Calpain, caspase-12 and procaspase-3 were analyzed by Western blotting and β-actin was
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used as an internal control. (G) Cells were pretreated with 30 µM 2-APB for 1 h and were
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then treated with 15 µM of compound K for 48 h. Procaspase-3 were analyzed by Western
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blotting and β-actin was used as an internal control.
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Fig. 5. Effects of 4-PBA on compound K induced ER stress, [Ca2+]i and apoptosis. (A) Cells
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were pretreated with 4 mM 4-PBA for 1 h and were then treated with 15 µM of compound K
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for 48 h. GRP78, IRE1α, p-eIF2α, m-calpain, caspase-12, and procaspase-3 were analyzed by
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Western blotting and β-actin was used as an internal control. (B) Effect of the 4-PBA on
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apoptosis was determined by testing the co-staining with PI and FITC-conjugated annexin V,
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and the translocation of phosphatidylserine (PS) was detected by flow cytometry. (C)
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Cytosolic Ca2+ levels were measured by fluo-3 AM fluorescence dye and were measured 26
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presented as means ± SD of three independent experiments. ***P < 0.001 vs. the compound
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