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 Ca2[.]
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 ACCEPTED MANUSCRIPT Compound K induced apoptosis via endoplasmic reticulum Ca2+ release through ryanodine receptor in human lung cancer cells Dong-Hyun Shin1, Dong-Gyu Leem1,3, Ji-Sun Shin1, Joo-Il Kim1, Kyung-Tack Kim2, Sang Yoon Choi2, Myung-Hee Lee2, Jung-Hye Choi3,4, Kyung-Tae Lee1,3* RI PT Seoul 130-701, Republic of Korea 10 11 Republic of Korea 12 13 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 14 Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea 15 * Corresponding author: Kyung-Tae Lee, PhD EP 16 M AN U Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, SC TE D Department of Pharmaceutical Biochemistry, College of Pharmacy 18 Kyung Hee University 19 Dongdaemun-Ku, Hoegi-Dong, Seoul, 130-701, 20 Republic of Korea 21 Tel.: +82 961 0860; Fax: +82 966 3885 22 E-mail : ktlee@khu.ac.kr AC C 17 23 24 Running title: Compound K induces apoptosis via ER Ca2+ release 25 ACCEPTED MANUSCRIPT Abstract 27 Background: Extended endoplasmic reticulum (ER) stress may initiate apoptotic pathways in 28 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 33 role of endoplasmic reticulum (ER) stress have not yet been understood 34 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 36 calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM 37 Results: Compound K-induced ER stress was confirmed through increased phosphorylation 38 of eIF2α and protein levels of GRP78/BiP, XBP-1S, and IRE1α in human lung cancer cells 39 Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m- 40 calpain activities that were both significantly inhibited by pretreatment either with BAPTA- 41 AM (an intracellular Ca2+ chelator) or dantrolene (a RyR channel antagonist) These results 42 were correlated with the outcome that compound K induced ER stress-related apoptosis 43 through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12) partially ameliorated 44 this effect Interestingly, 4-PBA (ER stress inhibitor) dramatically improved the compound K- 45 induced apoptosis 46 Conclusion: Cell survival and intracellular Ca2+ homeostasis during ER stress in human lung 47 cancer cells are important factors in the induction of the compound K-induced apoptotic 48 pathway AC C EP TE D M AN U SC RI PT 26 49 50 Keywords: Apoptosis, Calcium, Compound K, ER stress, Lung cancer cells ACCEPTED MANUSCRIPT Introduction 52 Apoptosis plays a pivotal role in the process of physiological cell deletion to maintain the 53 balance between cellular replication and death Some agents possessing chemotherapeutics 54 and chemoprevention properties have exhibited their ability by inducing either a cell cycle 55 arrest or apoptotic cell death Therefore, the induction of apoptosis is an attractive strategy for 56 treatment of cancer [1, 2] Apoptotic signaling can be initiated through two pathways: (i) the 57 extrinsic pathway via death receptors, such as, tumor necrosis factor receptor (TNFR1) and 58 FAS, which is activated by ligation of certain ligands Activation of death receptor results in 59 initiation of the caspase-8-dependent cell death process [3]; and (ii) an intrinsic pathway via 60 mitochondria Intrinsic apoptosis pathway is mediated by the release of cytochrome c from 61 mitochondria, which triggers the activation of caspase-9, cleaves caspase-3, and ultimately 62 promotes apoptotic cell death [4] Therefore, activation of caspases is a critical event in the 63 progressions of apoptosis M AN U SC RI PT 51 Endoplasmic reticulum (ER) is not only a reservoir for intracellular Ca2+, but also a home 65 for folding and posttranslational modifications of secretory proteins [5, 6] ER stress occurs 66 when ER is subjected to adverse situations such as hypoxia, nutrient deprivation, fails of 67 posttranslational modifications, imbalance in Ca2+ homeostasis, or an increased accumulation 68 of unfolded proteins [7] ER stress causes the unfolded protein response (UPR) to enhance the 69 protein folding capacity and decrease protein synthesis, and UPR activation from ER stress 70 initiates intracellular signaling pathways for cell protection EP AC C 71 TE D 64 The ER stress response is mainly regulated by three ER transmembrane proteins: activating 72 transcription factor-6 (ATF-6), inositol requiring kinase-1 (IRE-1), and protein kinase-like 73 endoplasmic reticulum kinase (PERK) [8] Under usual conditions, these transmembrane 74 proteins keep inactive bound with ER-resident chaperone glucose-regulated protein-78 75 (GRP78/BiP) The UPR is regulated by an ER stress sensor IRE1α [9] Activation of IRE1α, a ACCEPTED MANUSCRIPT serine/threonine protein kinase/endoribonuclease, leads to the splicing of X-box binding 77 protein-1 (XBP-1) mRNA This process encodes a transcriptional activator, which initiates 78 the transcription of chaperone protein-encoding genes, whose products have a function of ER 79 protein folding [10] PERK, another ER stress transducer, is a transmembrane kinase that 80 phosphorylates eukaryotic translation initiation factor subunit α (eIF2α), resulting in 81 reduction of protein synthesis and counteraction of ER protein overload [11] Active 82 fragments of ATF-6 translocate into the nucleus and bind to the ER stress response element 83 thereby stimulating transcription of transcription factor genes (e.g XBP-1) and ER chaperone 84 genes (e.g GRP78) as well as expression of CCAAT/enhancer-binding protein-homologous 85 protein (CHOP/GADD153) [7] Severe or sustained ER stress can provokes pro-apoptotic 86 ATF-6, PERK, and IRE-1 signaling and induces CHOP increase Furthermore, caspase-12 87 participates in ER stress-mediated apoptosis, as shown by the resistance to ER stress-induced 88 apoptosis in caspase-12 knockdown mice [12] SC M AN U The intracellular Ca2+ level handles a various cellular phenomenon including transcription, TE D 89 RI PT 76 exocytosis, apoptosis, and proliferation [13], and its concentration is under the control of ion 91 channels, multiple pumps, and binding proteins [14] The Ca2+ release from the ER is 92 mediated through the ryanodine receptors (RyRs) and ER-resident inositol trisphosphate 93 receptors (IP3Rs) [15] However, even with tight regulation of Ca2+ release from the ER, the 94 exhaustion of ER Ca2+ and the overload of cytosolic Ca2+ can occur due to several stimuli 95 The disrupted Ca2+ homeostasis and unchecked increases in cytosolic Ca2+ can induce 96 apoptosis via the activation of ER resident caspases and the activation of processes in the 97 cytoplasm [16-18] 98 99 100 AC C EP 90 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 ACCEPTED MANUSCRIPT reported to possess a variety of biological properties including neuroprotective, anticancer 102 and anti-inflammatory activities [19] The two major sub-types of ginsenosides have been 103 termed protopanaxadiols and protopanaxatriols, which after ingestion can give rise to novel 104 metabolites in the body [16, 17] 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol 105 (compound K) (Fig 1A) is a major metabolite of several protopanaxadiol-type ginsenosides, 106 including Rb1, Rb2 and Rc, by intestinal bacteria through the multistage cleavage of sugar 107 moieties [20] It has been investigated in terms of its anti-diabetic [21-23], anti-inflammatory 108 [24-26] and anticancer [27, 28] effects 109 Although a previous study suggested a role for ER stress in the apoptosis in colon cancer cells 110 [29], the role of Ca2+ released from ER and caspase-12 in compound K-induced apoptosis in 111 human lung cancer cells has not yet been reported We report that compound K induces Ca2+ 112 release from ER via the RyR channel, leading to the activation of m-calpain, caspase-12 and 113 caspase-3 In addition, there is an increase in the levels of the endoplasmic reticular stress 114 markers, IRE1α, XBP-1S and GRP78 AC C EP TE D M AN U SC RI PT 101 ACCEPTED MANUSCRIPT 115 Materials and methods 116 118 2.1 Chemicals and reagents Compound K (purity >98%) used in present study was isolated from P ginseng, and its RI PT 117 structural identities were determined spectroscopically (1H and 13NMR, IR, MS) as 120 previously described (25) RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS), 121 penicillin, streptomycin and Fura-2/AM were obtained from Life Technologies Inc 3-(4,5- 122 Dimethylthiazol-2-yl)- 2,5-diphenyl-tertazolium bromide (MTT), dimethyl sulfoxide 123 (DMSO), RNase A, phenylmethylsulfonylfluoride (PMSF), propidium iodide (PI), 4- 124 phenylbutyrate (4-PBA), 2-Aminoethoxydiphenyl Borate (2-APB), Dantrolene, BAPTA-AM, 125 ethylene glycol tetraacetic acid (EGTA) and CGP37157 were purchased from Sigma 126 Chemical Co The following antibodies for caspase-3, caspase-7, caspase-9, poly (ADP- 127 ribose) polymerase (PARP), XBP-1, c-FLIPL, Bid, Bcl-2, Bcl-xL, p-eIF2α, GADD 153, m- 128 calpain and β-actin were purchased from Santa Cruz Biotechnology, Inc The antibodies for 129 X-linked inhibitor of apoptosis protein (XIAP), caspase-8 and caspase-12 were purchased 130 from BD Biosciences, Pharmingen The antibody for IRE1α and GRP78 were purchased 131 from Cell Signaling Technology z-VAD-fmk, z-ATAD-fmk were purchased from 132 Calbiochem 134 135 M AN U TE D EP AC C 133 SC 119 2.2 Cell culture and sample treatment Human lung adenocarcinoma A549 and human lung squamous cell carcinoma SK-MES- 136 were purchased from the Korea Cell Line Bank (Seoul, Republic of Korea) A549 was 137 grown in RPMI-1640 medium with 10% FBS, penicillin (100 units/ml) and streptomycin 138 sulfate (100 µg/ml) in a humidified atmosphere (5% CO2, 95% humidity, 37°C) SK-MES-1 139 was grown in DMEM medium with 10% FBS, penicillin (100 units/ml) and streptomycin ACCEPTED MANUSCRIPT 140 sulfate (100 µg/ml) in a humidified atmosphere (5% CO2, 95% humidity, 37°C) Cells were 141 incubated for 48 h with various concentrations (5, 10, 15 µM) or with 15 µM of compound K 142 for various times (6, 12, 24, 36, or 48 h) in 2% FBS contained medium 144 145 RI PT 143 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- 147 well plates Then, cells were treated with various concentrations of compound K and 148 incubated for 48 h After 48 h, 20 µl of MTT [5 mg/ml stock solution in phosphate-buffered 149 saline (PBS)] were added and further incubated for h The medium was removed and 150 formazan blue formed in the cells was dissolved with dimethyl sulfoxide (200 µl) The 151 optical density was measured at 540 nm M AN U SC 146 152 154 2.4 Quantification of apoptosis via PI and Annexin V double staining TE D 153 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 156 conducted with µl of PI (50 µg/ml) and µl of FITC-conjugated Annexin V for 30 at 157 room temperature in dark place Thereafter, 400 µl of binding buffer were added for analysis 158 via fluorescence-activated cell sorting (FACS) cater-plus flow cytometry (Becton Dickinson 159 Co, Heidelberg, Germany) 161 162 AC C 160 EP 155 2.5 Preparation of cellular protein and Western blot analysis Cells treated with Compound K were washed twice with ice-cold PBS and were 163 centrifuged at 200 g for The obtained cell pellet was suspended in PRO-PREPTM 164 protein extraction buffer (Intron, Seoul, Republic of Korea) Equal amounts of protiens were ACCEPTED MANUSCRIPT separated via SDS-PAGE and were transferred to PVDF membranes for Western blot 166 analysis The immunoblot was incubated specific primary antibodies overnight at 4°C HRP- 167 conjugated secondary antibodies were detected using an enhanced chemiluminescence 168 detection system (GE healthcare, WI, USA) RI PT 165 169 171 2.6 Calcium quantification A549 and SK-MES-1 cells grown on cover glass were incubated overnight Cytosolic free SC 170 Ca2+ was measured using the Ca2+-sensitive fluorescent indicator dye Fura-2/AM Cells 173 grown on a matrigel-coated cover-slide bottom dish were washed three times with PBS and 174 were incubated in the dark for 30 at room temperature with Fura-2/AM (final 175 concentration µM) in PBS The cells were washed again with PBS three times and were 176 analyzed by being illuminated with an alternating light of 340 and 380 nm from a rotating 177 filter wheel M AN U 172 180 2.7 Detection of calcium concentration Free cytosolic calcium was measured by a fluorescence Ca2+ indicator Fluo-3/AM Treated EP 179 TE D 178 cells were washed twice with HBSS (130 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 10 mM 182 HEPES, 10 mM glucose, mM CaCl2, pH 7.4), re-suspended, and then incubated with the 183 Fluo-3/AM (3 mM) for 30 The free cytosolic Ca2+ levels were analyzed by flow 184 cytometry (Becton Dickinson Co, Heidelberg, Germany) 185 186 AC C 181 2.8 Statistical analysis 187 The results are expressed as the mean ± S.D of triplicate experiments Statistically 188 significant values were compared using ANOVA and Dunnett’s post hoc test Statistical ACCEPTED MANUSCRIPT 189 analysis was performed using SigmaPlot software version 10.0 (Systat Software, Inc., San 190 Jose, CA, USA) P-values of less than 0.05 were considered to be statistically significant 191 AC C EP TE D M AN U SC RI PT 192 ... MANUSCRIPT Compound K induced apoptosis via endoplasmic reticulum Ca2+ release through ryanodine receptor in human lung cancer cells Dong-Hyun Shin1, Dong-Gyu Leem1,3, Ji-Sun Shin1, Joo-Il Kim1, Kyung-Tack... 3.1 Compound K induced caspase-dependent apoptosis in human lung cancer cells 195 We examined the effect of compound K on the cell viabilities using MTT assays in human lung cancer cells Compound. .. z-ATAD-fmk partially inhibited 248 compound K- induced apoptosis, which implied that compound K induced apoptosis in 249 human lung cancer cells via both caspase-12 and extrinsic and/or intrinsic