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DOI:10.1111/bph.13408 www.brjpharmacol.org

British Journal of Pharmacology

RESEARCH PAPER

Correspondence

Decursin enhances TRAILinduced apoptosis through oxidative stress mediatedendoplasmic reticulum stress signalling in non-small cell lung cancers

Sung-Hoon Kim, Cancer Preventive Material Development Research Center, College of Korean Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 131-701, South Korea. E-mail: [email protected]. † These authors equally contributed to this work. ---------------------------------------------------------

Received 10 October 2014

Revised 25 November 2015

Accepted 3 December 2015

Jaekwang Kim†, Miyong Yun†, Eun-Ok Kim, Deok-Beom Jung, Gunho Won, Bonglee Kim, Ji Hoon Jung and Sung-Hoon Kim College of Korean Medicine, Kyung Hee University, Seoul South Korea

BACKGROUND AND PURPOSE The TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. However, because most tumours exhibit resistance to TRAIL-induced apoptosis, the development of combination therapies to overcome resistance to TRAIL is required for effective cancer therapy.

EXPERIMENTAL APPROACH Cell viability and possible synergy between the plant pyranocoumarin decursin and TRAIL was measured by MTT assay and CalcuSyn software. Reactive oxygen species (ROS) and apoptosis were measured using dichlorodihydrofluorescein and annexin/propidium iodide in cell flow cytometry. Changes in protein levels were assessed with Western blotting.

KEY RESULTS Combining decursin and TRAIL markedly decreased cell viability and increased apoptosis in TRAIL-resistant non-small-cell lung cancer (NSCLC) cell lines. Decursin induced expression of the death receptor 5 (DR5). Inhibition of DR5 attenuated apoptotic cell death in decursin + TRAIL treated NSCLC cell lines. Interestingly, induction of DR5 and CCAAT/enhancer-binding protein homologues protein by decursin was mediated through selective induction of the pancreatic endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) branch of the endoplasmic reticulum stress response pathway. Furthermore, enhancement of PERK/ATF4 signalling by decursin was mediated by ROS generation in NSCLC cell lines, but not in normal human lung cells. Decursin also markedly down-regulated expression of survivin and Bcl-xL in TRAIL-resistant NSCLC cells.

CONCLUSIONS AND IMPLICATIONS ROS generation by decursin selectively activated the PERK/ATF4 axis of the endoplasmic reticulum stress signalling pathway, leading to enhanced TRAIL sensitivity in TRAIL-resistant NSCLC cell lines, partly via up-regulation of DR5.

Abbreviations ATF, activating transcription factor; BiP, polypeptide binding protein; CHOP, CCAAT/enhancer-binding protein homologues protein; c-IAP, cellular inhibitor of apoptosis protein; DcR, decoy receptor; DR, death receptor; eIF2, eukaryotic initiation factor 2.; ER, endoplasmic reticulum; IRE1, inositol-requiring kinase 1; NSCLC, non-small-cell lung cancer; PDI, protein disulfide isomerase; PERK, protein kinase RNA-like endoplasmic reticulum kinase; ROS, reactive oxygen species; TRAIL, TNF-related apoptosis-inducing ligand; UPR, unfolded protein response; XIAP, X-linked inhibitor of apoptosis

© 2015 The British Pharmacological Society

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Tables of Links TARGETS Enzymes

LIGANDS

a

Other proteins

IRE-1α

Bcl-2

PERK

c-IAP1

Catalytic receptors

b

c

TNFα TRAIL

c-IAP2

DcR1, decoy receptor 1

Survivin

DcR2, decoy receptor 2

XIAP

DR4, death receptor 4 DR5, death receptor 5

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide a,b,c Alexander et al., 2015a,b,c). to PHARMACOLOGY 2015/16 (

Introduction Non-small-cell lung cancer (NSCLC) is the main cause of cancer-related deaths worldwide (Jemal et al., 2011). In spite of continuous efforts to develop effective molecular-targeted or combinational therapies, treatment for NSCLC remains sub-optimal due to the development of resistance to therapies. New therapeutic strategies to overcome resistance are urgently needed to improve the effectiveness of NSCLC treatment. As the ability of malignant cells to evade apoptosis is a characteristic feature of cancer and cellular resistance to apoptosis constitutes an important clinical problem (Jia et al., 2012; Lopez-Beltran et al., 2007), apoptosis has recently received much attention in cancer research. Apoptosis is primarily induced via the ‘intrinsic pathway’, which is typically activated by endogenous stresses, such as DNA damage, hypoxia or other cell stresses; and the ‘extrinsic pathway’, which is mediated by cell surface death receptors such as the TNF receptor superfamily (Declercq et al., 2011; Hellwig et al., 2010; Holoch and Griffith, 2009). In humans, one member of the TNF superfamily, TNF-related apoptosis-inducing ligand (TRAIL), interacts with four known membrane-bound receptors: two death receptors -DR4/TRAIL receptor-1 and DR5/TRAIL receptor-2 - and two decoy receptors -DcR1/TRAIL receptor-3 and DcR2/TRAIL receptor-4. The decoy receptors have a truncated cytoplasmic death domain (Pan et al., 1997a; Pan et al., 1997b). Activation of TRAIL receptor-mediated apoptosis is an attractive tumour therapy for specifically killing cancer cells, leaving normal cells unaffected (Walczak et al., 1999). However, the fact that many types of cancer – including NSCLS – exhibit TRAIL resistance limits the clinical utility of TRAIL. The endoplasmic reticulum (ER) controls protein folding and assembly into multi-subunit complexes for extracellular secretion (Yewdell and Nicchitta, 2006). Generation of misfolded or unfolded proteins due to various stresses, such as the formation of reactive oxygen species (ROS), is corrected by activation of the unfolded protein response (UPR) (van der Vlies et al., 2003; Yewdell and Nicchitta, 2006). UPR signalling is mediated by three major transducers: the inositol-requiring kinase 1 (IRE1), the eukaryotic translation initiation factor-2α kinase 3 (PERK) and activating transcription factor 6 (ATF6) (Schroder 1034

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and Kaufman, 2005; Van Anken and Braakman, 2005). The polypeptide binding protein (BiP or GRP78) is a master protein that recognizes misfolded proteins in the ER. The interaction of GRP78 with misfolded proteins results in activation of the three UPR signal transducers (Patil and Walter, 2001). GRP78 increases the phosphorylation of eukaryotic initiation factor 2 (eIF2) at Ser51 by inducing the dimerization of PERK. Phosphorylated eIF2 can attenuate translational initiation (Shi et al., 1998). In contrast, eIF2 phosphorylation induces translation of activating transcription factor 4 (ATF4) mRNA. The PERK/eIF2/ATF4 regulatory axis also induces expression of oxidative stress response genes and of those encoding proteins with pro-apoptotic functions, such as CCAAT/enhancer-binding protein homologues protein (CHOP), which can induce DR4 and DR5 (Wek and Cavener, 2007; Xu et al., 2012; Yamaguchi and Wang, 2004). In the current study, we focused on the mechanism by which the plant pyranocoumarin, decursin, already known to exhibit anti-tumour activity in vitro (see Zhang et al., 2012) sensitized TRAIL-resistant human NSCLC cells to TRAIL-mediated apoptosis and investigated the involvement of ROS-mediated induction of selective ER stress signals.

Methods Cell culture Human NSCLC cells (A549, H596, H1299 and Calu-1) and BEAS-2B normal bronchial epithelial cells (ATCC® CRL-9609™ ) were obtained from the American Type Culture Collection (ATCC, USA) and grown in RPMI 1640 medium containing 10% heat-inactivated FBS and 1% antibiotics (Welgene, South Korea) in a humidified atmosphere of 95% air and 5% CO2 at 37°C.

Cell viability assay The cytotoxicity of decursin and TRAIL, alone or in combination, was evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, MO, USA) assay. Cells were seeded in 96-well microplates at a density of 1 × 104 per and treated with various concentrations of decursin. After incubation for the

Decursin induces ROS dependent ER stress for TRAIL-induced apoptosis indicated time, MTT (1 mgmL 1) was added, followed by a further 2 h incubation. OD at 570 nm was measured using a microplate reader (Tecan Austria GmbH, Grödig, Austria). MTT assay was carried out in triplicate and also was repeated separately three times.

Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA(2 μg) isolated from NSCLC cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was reverse transcribed and then subjected to PCR by using GeneAmpPCR 2720 (Applied Biosystems, Foster City, CA, USA). Reverse transcription was carried out using oligo(dT)18 primer and M-MLV reverse transcriptase (Invitrogen Life Technologies) at 37°C for 50 min, a thermal program of 50°C for 60 min and 85°C for 5 min. The cDNAwas amplified by PCR using the following specific primers: DR4 (forward: 5′-GGCTGAGGACAATGCTC ACA-3′, reverse: 5′-TTGCTGCTCAGAGACGAAAGTG-3′), DR5 (forward: 5′-GACTCTGAGACAGTGCTTCGATGA-3′, reverse: 5′-CCATGAGGCCCAACTTCCT-3′), CHOP (forward: 5′-CAACT GCAGAGAATTCAGCTGA -3′, reverse: 5′-ACTGATGCTCTAGAT TGTTCAT-3′), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward: 5′-CCACTCCTCCACCTTTGAC-3′, reverse: 5′-ACCCTGTTGCTGTAGCCA-3′ (Bioneer, Korea). A total of 30 cycles were carried out of denaturation for 15 s at 94°C, annealing for 0.5 min at 60°C and extension for 1 min at 72°C, followed by incubation for an additional 5 min at 72°C. The amplified products were analysed by electrophoresis with 1.5% agarose gel and visualized using ethidium bromide staining under the image capture and analysis system of ChemiGenius (Syngene). Data were obtained from three separate experiments.

Real-time quantitative PCR (RT-qPCR) RT-qPCR was performed to quantify mRNA expression of CHOP, DR5 and DR4 in the experimental groups using a LightCycler™ instrument (Roche Applied Sciences, IN, USA) according to the manufacturer’s protocol. The mRNA expression of GAPDH was used to normalize the expression of genes of interest. A549 and H596 cells were treated by decursin for 48 h. Total RNA was isolated using the RNA easy kit (Invitrogen). First-strand cDNA was synthesized from 500 ng of total RNA using a PrimeScript® reverse transcriptase (TaKaRa, Japan). PCR conditions were 95°C for 30 s followed by 40 cycles at 95°C for 5 s and 60°C for 34 s. GAPDH was used as an internal reference gene to normalize the expression of apoptotic genes. Relative quantification of apoptosis-related genes was analysed by the comparative threshold cycle method. Data were obtained from three separate experiments.

Western blotting Total protein was extracted from A549, H596, H1299 and Calu-1 cells using RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% deoxycholic acid-Na, 1 M EDTA, 1 mM Na3VO4, 1 mM NaF) containing a protease inhibitor cocktail (Roche). Samples (30 μg) were quantified using a Bio-Rad DC protein assay kit II (Bio-Rad, Hercules, CA, USA), separated by electrophoresis on an 8–12% SDSPAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories). After blocking with 1–5% non-fat skim milk, the membrane was probed with antibodies for CHOP, DR5,

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ATF4, GRP78, Calnexin, PERK, protein disulfide isomerase (PDI), IRE-1α (Cell Signaling, Beverly, MA, USA) and DR4, PARP, cleaved caspase 3, caspase 3, cleaved caspase 8, caspase8, survivin, cellular inhibitor of apoptosis protein 1 (c-IAP1) and c-IAP2 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA). Monoclonal anti-β-actin was purchased from SigmaAldrich (St. Louis, MO, USA). Antibodies to DcR1, DcR2 and the X-linked inhibitor of apoptosis (XIAP) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Secondary antibodies coupled to horseradish peroxidase were from Vector Labs (Burlingame, CA, USA). Expression was visualized using enhanced chemiluminescence Western blotting detection reagent (Amersham Pharmacia, Piscataway, NJ, USA). Data were obtained from three separate experiments.

Immunofluorescence assay Cells were plated onto covered glass-bottom dishes (SPL Life Sciences, Seoul, South Korea). Cells were fixed in 4% paraformaldehyde for 10 min at room temperature and then permeabilized in 0.5% Triton X-100 in PBS for 5 min at room temperature. Cells were labelled with primary antibody diluted in PBS (1:500) overnight at 4°C, and then with secondary Alexa Red-conjugated antibodies (Abcam, Cambridge, UK) diluted 1:100 with 1% BSA/PBS for 2 h at room temperature. The samples were mounted with mounting medium containing DAPI and visualized using an Olympus FLUOVIEW FV10i (Olympus, Tokyo, Japan) confocal microscope.

Apoptosis assay NSCLCs were seeded in six-well plates, treated with the indicated compounds for 24 h and stained with an Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) kit (Bio Vision Technology Inc., Golden, CO), then analysed using FACS. rmTRAIL R2/TNFRSF10B Fc Chimera (R&D Systems, Inc., MN, USA) was used as a DR5 antagonist; it was added as a co-treatment with decursin and TRAIL for 24 h.

ROS measurement Hydrogen peroxide formation was analysed using dichlorodihydrofluorescein diacetate (H DCFDA) (Invitrogen). Cells were equilibrated with 1 μM DCFDA for 1 h and then resuspended in fresh medium. DCFDA was analysed using FACS. Data were obtained from three separate experiments.

siRNA transfection Transfection of control vector or DR5 siRNA or catalase/ pCMV6A (Bioneer, Seoul, Korea) was performed using INTERFERin transfection reagent (Polyplus-transfection INC., New York, NY) according to the manufacturer’s protocol.

Validation of synergy between decursin and TRAIL To determine the synergy between decursin and TRAIL, cytotoxicity assays were performed in which the concentrations of decursin and TRAIL were gradually increased, while their ratio was maintained constant. The results were analysed by the CalcuSyn software (Biosoft, MO, USA). The fraction of living cells at each concentration was used for the analysis of synergism between decursin and TRAIL by the CalcuSyn software. British Journal of Pharmacology (2016) 173 1033–1044

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Data analysis Data are presented as means ± SD. Statistically significant differences between control and decursin-treated groups were calculated by Student’s t-test. All experiments were carried out at least three times.

Materials Decursin was purchased from Sigma. Recombinant human TRAIL/Apo2L was purchased from Atgen (Gyeonggi-do, South Korea).

Results Combination treatment with decursin and TRAIL synergistically induces cytotoxicity in TRAIL-resistant NSCLC cells To examine their sensitivity to TRAIL, NSCLC cell lines were incubated with various concentrations of TRAIL. Consistent with previous reports (Jin et al., 2007), A549, H596, H1299 and Calu-1 cells showed strong resistance to TRAIL, while

H460 cells were more sensitive, exhibiting an IC50 of 62 ngmL 1 (Supporting Information Fig. S1). In contrast, decursin was similarly cytotoxic to all cells, with IC50 values of approximately 100–200 μM (Figure 1A). Treatment with a fixed conccnetration of decursin (150 μM) for different times revealed that viability of NSCLC cells decreased in a time-dependent manner (Figure 1B). Interestingly, cotreatment with decursin and TRAIL was found to synergistically decrease the viability of NSCLC cells (Figure 1C). Surprisingly, the combination index (CI) values were below 0.8 at almost all fraction-affected points, implying strong synergy in all four tested TRAIL-resistant cell lines (Figure 1D and Supporting Information Fig. S2) including H460, which is TRAIL sensitive (Supporting Information Fig. S3A and B).

Combination treatment with decursin and TRAIL induces apoptosis in TRAIL-resistant NSCLC cells The major cell death mechanism through which TRAIL receptors act is known as the ‘extrinsic pathway’ of apoptosis, which is induced by an extracellular death signal (Wallach

Figure 1 Synergistic cytotoxic effect of decursin and TRAIL co-treatment in TRAIL-resistant lung cancer cell lines. (A) Four NSCLC cell lines were treated with the indicated concentrations of decursin for 24 h; cell viability was then analysed using an MTT assay. (B) Cytotoxicity of decursin (150 μM) in 1 NSCLC cells was measured at the indicated times. (C) Cells were co-treated with the indicated concentrations of decursin and 20 ngmL TRAIL for 24 h. Cytotoxicity was then analysed using an MTT assay. Data are shown as means ± SD from three separate experiments. Also, each experiment was performed in triplicate. (D) Combination index (CI) values with fraction affected (Fa) between decursin and TRAIL in NSCLC cells, calculated using the CalcuSyn software. 1036

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Decursin induces ROS dependent ER stress for TRAIL-induced apoptosis

et al., 1999). To examine whether decursin overcomes the TRAIL resistance due to inhibition of death signalling through receptors, we analysed apoptosis in cells treated with a combination of decursin and TRAIL. Cleaved caspase 3, cleaved caspase 8 and cleaved PARP levels were dramatically increased by the combination of decursin and TRAIL. The concentrations used for co-treatment were half those of each agent alone (Figure 2A). Consistent with these findings, FACS analysis with Annexin V and PI revealed that the decursin /TRAIL combination treatment enhanced considerably the apoptotic population, compared with untreated or single-agent-treated A549 and H596 cells (Figure 2B), demonstrating that decursin drives TRAIL-resistant cells to undergo apoptosis.

Decursin induces DR5, but not DR4, in TRAIL-resistant NSCLC cells Accumulating evidence reveals that resistance of cancer cells to TRAIL is acquired through various mechanisms, such as suppression of DR4 and/or DR5 and induction of antiapoptotic proteins (Yamaguchi and Wang, 2004). To elucidate the mechanism by which decursin overcomes TRAIL resistance, we evaluated the expression of the death receptors, DR4 and DR5, and the decoy receptors, DcR1 and DcR2, along with that of anti-apoptotic proteins that block extrinsic

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apoptosis signalling. In A549 and H596 cells, decursin treatment increased DR5 expression, while the expression levels of other death receptors – including DR4, DcR1 and DcR2 – were unchanged compared with untreated controls (Figure3A). Additionally, decursin did not affect the protein levels of anti-apoptotic molecules such as XIAP, c-IAP1 and c-IAP2 under identical conditions (Figure 3A). DR5 expression was consistently induced by decursin in a time-dependent and dose-dependent manner (Figure 3B and C). As the endogenous expression level of membraneembedded death receptors can vary due to the activity of endocytotic mechanisms (Zhang and Zhang, 2008), DR5 cell surface expression was examined by FACS. In three NSCLC cell lines, decursin treatment resulted in a more than twofold increase of DR5 on the cell membrane, compared with the untreated control, while DR4 expression was unchanged (Figure 3D). These data indicate that decursin markedly increased expression of DR5, but not DR4 or other death receptors or anti-apoptotic proteins, in NSCLC cells.

Inhibition of DR5 attenuates decursin-induced apoptosis To determine whether DR5 was an important mediator of decursin-induced apoptosis, we used knockdown of DR5

Figure 2 1

Synergistic apoptotic effect of decursin and TRAIL co-treatment in NSCLC cells. (A) Cells were treated with 30 μM decursin (+), 10 ngmL TRAIL 1 (+), 60 μM decursin (++) or 20 ngmL TRAIL (++) alone or combination decursin (+)/TRAIL (+) or decursin (++)/TRAIL (++)) for 24 h. Cell lysates were prepared and subjected to Western blotting with antibodies for PARP, caspase 8, cleaved caspase 8 (44/42) (C-caspase 8 (44/42)), cleaved caspase 8 (13) (C-caspase 8 (13)), caspase 3 and cleaved caspase 3 (C-caspase 3); β-actin was used as an internal standard. A representative blot of 1 three separate experiments. (B) Cells were co-treated with 60 μM decursin and 20 ngmL TRAIL as described previously. After staining with Annexin V-FITC and PI, apoptotic cells were analysed using a flow cytometer. The numbers in each plot indicate percentages of apoptotic cells. Annexin V assay was repeated three times. British Journal of Pharmacology (2016) 173 1033–1044

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Figure 3 Effect of decursin on expression of death receptors, decoy receptors and anti-apoptotic proteins in TRAIL-resistant lung cancer cells. (A) Protein 1 expression in A549 and H596 cancer cells treated with decursin (60 μM), TRAIL (20 ngmL ), or with their combination. Total cell protein extracts were subjected to immunoblotting with the indicated antibodies. (B) A549 and H1299 cells were treated with decursin (60 μM) for the indicated 5 times and subjected to immunoblotting as described previously. (C) A549 and H596 (3 × 10 cells per well) were treated with 0, 30, 60 or 120 μM of decursin for 24 h; whole-cell extracts were then prepared and analysed by Western blotting. A representative blot of three separate experiments. (D) A549, H596 and H1299 cells were treated with 60 μM decursin for 24 h. Cells were then stained with antibodies for DR5, DR4 (green; FITC-labelled primary antibodies), or an IgG control (green; FITC-labelled antibody) and analysed for cell surface expression of DR4 and DR5 by flow cytometry. A representative blot of three separate experiments.

by transfection with siRNA for DR5. In the H1299 NSCLC cell line, the cytotoxicity of the combination of decursin and TRAIL was clearly attenuated, after DR5 knockdown (Figure 4A). Similarly, Annexin V/PI FACS analysis revealed that, following siRNA for DR5, the decursin-induced apoptosis was decreased (Figure 4B). Moreover, siRNA-mediated silencing of DR5 attenuated the cleavage of PARP, cleaved caspase 8 and caspase 3 induced by decursin (Figure 4C). These data suggest that DR5 induction is one of important mechanisms by which decursin mediated apoptosis in TRAILresistant NSCLC cells.

Decursin selectively induces the ATF4/CHOP axis of three major ER stress signals, leading to up-regulation of DR5 CHOP is a well-known transcriptional activator of DR5 (Lee et al., 2012; Oh et al., 2010). We therefore assessed the expression of CHOP in decursin-treated cells. RT-PCR and real-time quantitative PCR revealed that CHOP and DR5, but not DR4, were dose-dependently up-regulated by decursin at the mRNA level in A549 and H596 cells (Figure 5A and B). CHOP and DR5 were also significantly induced at the protein level in A549, H596, H1299 and Calu-1 cells (Figure 5C). CHOP induction is a common response to ER stress following unfolded protein accumulation (Yamaguchi and Wang, 2004). To examine the involvement of the ER stress response, levels 1038

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of proteins involved in the ER stress response were measured after treatment of NSCLC cells with decursin. Decursin activated only certain components of PERK/ATF4 axis signalling, such as GRP78, PERK and ATF4, as well as the downstream targets CHOP and DR5, whereas other ER stress signalling components – such as ATF6 and IRE-1a, as well as chaperones – were unaffected (Figure 5D). In a more detailed assay of Xbp1 splicing in various cell lines and time points, we found that decursin did not induce Xbp1 splicing, while treatment with thapsigargin, an ER stress inducer, increased the spliced Xbp1 level dramatically (Figure 5E and Supporting Information Fig. S4). Immunofluorescence assays confirmed that expression of ATF4 in the nucleus was dramatically increased in decursin-treated cells compared with controls (Figure 5F). Accordingly, siRNA-mediated silencing of ATF4 abolished decursin-mediated CHOP and DR5 induction, but not that of the ATF4 upstream molecule PERK (Figure 5G). Taken together, these findings demonstrate that the up-regulation of DR5/CHOP by decursin was mediated by selective activation of the PERK/ATF4 signalling components of three major ER stress signal pathways.

ROS generated by decursin selectively activates ATF/PERK signalling Specific activation of ATF/PERK signalling by decursin prompts the question of which molecules participate in the

Decursin induces ROS dependent ER stress for TRAIL-induced apoptosis

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Figure 4 Effect of DR5 inhibition on decursin-induced/TRAIL-induced apoptosis in DR5 siRNA-transfected H2199 cells. (A) DR5 depleted or intact H1299 1 cells were exposed to 60 μM decursin and 20 ngmL TRAIL for 24 h. Cell viability was measured using an MTT assay. Con: control, D: decursin, T: TRAIL, DT: decursin + TRAIL. *** = P < 0.001, ** = P < 0.01, * = P < 0.05. MTT assay was conducted in triplicate, in three separate experiments. 1 (B) 60 μM decursin and 20 ngmL TRAIL were treated in DR5 depleted or intact H1299 cells for 24 h and apoptotic cells were analysed using a flow cytometer after staining with Annexin V-FITC and PI. Con: control, D: decursin, T: TRAIL, DT: decursin + TRAIL. A representative blot of three separate experiments. (C) H1299 cells were transfected with siRNA plasmid against DR5 or a control. After 48 h culture, the cells were treated with 1 60 μM decursin and 20 ngmL TRAIL for 24 h. Cell lysates were prepared and subjected to Western blotting with antibodies for PARP, caspase 8, cleaved caspase 8 (44/42) (C-caspase 8 (44/42)), cleaved caspase 8 (13) (C-caspase 8 (13)), caspase 3 and cleaved caspase 3 (C-caspase 3); β-actin was used as an internal standard. A representative blot of three separate experiments.

selective induction of ER stress signals. Previous reports have shown that ER stress signals can be induced by ROS and changes in redox potential (Guan et al., 2009; Malhotra et al., 2008). To examine whether decursin treatment induced ROS in TRAIL-resistant cancer cells, ROS were quantified by FACS. As shown in Figure 6A and B, treatment with decursin resulted in considerable ROS production by A549 and H1299 cells in a dose-dependent and time-dependent manner (Figure 6A and B, and Supporting Information Fig. S5A and B). However, ROS generation was not stimulated by decursin in BEAS-2B immortalized normal bronchial epithelial cells (Supporting Information Fig. S6), but other cancer cells showed more than twofold up-regulation of ROS in response to decursin, compared with untreated controls (Figure 6C), indicating thetherapeutic potential of decursin for NSCLC treatment.

To evaluate whether selective induction of ATF4/PERK was dependent on ROS generation, cells were treated with three specific ROS scavengers, sodium pyruvate (SP), N-acetyl cysteine (NAC) or dimethylthiourea (DMTU). Treatment with SP attenuated the induction of GRP78, ATF4, CHOP and DR5 by decursin (Figure 6D). Likewise, the other scavengers attenuated DR5 induction by decursin (Supporting Information Fig. S7A and B). We also confirmed that ROS production was reduced in catalase-transfected A549 cells more than in intact A549 control cells (Figure 6F). In addition, SP treatment markedly decreased apoptosis induced by cotreatment with decursin and TRAIL in A549 and H1299 cells (Figure 6E). These data indicated that CHOP/DR5 induction by decursin was mediated by ROS generation, which was responsible for the selective activation of the ATF4/PERK axis of ER stress signalling. British Journal of Pharmacology (2016) 173 1033–1044

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Figure 5 Decursin-induced CHOP/DR5 up-regulation is mediated through selective induction of PERK/ATF4 ER stress signalling. (A and B) A549, H596 and H1299 cells were treated with 0, 30, 60 and 120 μM decursin (Dec) for 24 h. Total RNA was isolated, and (A) RT-PCR or (B) real-time qPCR analysis was performed. (C and D) NSCLC cells were treated with 60 μM decursin for 12 h, then analysed by Western blotting. (E) Decursin does not affect XBP-1 signalling. Cells were treated with decursin for 24 h or thapsigargin (TG) for 8 h. Cells were lysed, total RNA was isolated and RT-PCR was performed to detect the spliced and unspliced forms of XBP-1. PCR products were separated on a 3% agarose gel. USF; unspliced form, SF; spliced form. (F) H1299 cells were treated with 60 μM decursin for 24 h, and nuclear ATF4 expression was visualized using immunofluorescence. Red = ATF4; Blue = DAPI staining for nuclear DNA; Scale bar: 50 μm. A representative blot of three separate experiments. (G) A549 cells were transfected with siRNA plasmids against ATF4 or control. After 48 h, cells were treated with 60 μM decursin for 24 h, and whole-cell extracts were analysed by Western blotting. A representative blot of three separate experiments.

Discussion The use of TRAIL-based therapy for cancer is considered to be one of the most promising therapeutic strategies. However, many tumours acquire resistance to TRAIL-mediated cell death by induction of anti-apoptosis-related proteins such as decoy receptors (DcR1 and DcR2) (Sanlioglu et al., 2005; Wu et al., 2004), anti-apoptotic proteins (c-FLIP, IAPs, Bcl-2, Bcl-xL and Mcl-1) (Gillissen et al., 2010; Johnson et al., 2003; Zang et al., 2014) and survival proteins (PI3K, AKT and NF-κB) (Chen et al., 2001; Oya et al., 2001; Rychahou et al., 2005). To overcome the acquired resistance to TRAIL in cancers, alternative approaches that combine TRAIL with other classical agents to either enhance TRAIL activity or sensitize TRAIL-resistant cells have been utilized. Several natural compounds, such as curcumin, kurarinone and cryptotanshinone, can increase the TRAIL sensitivity of several TRAIL-resistant cancer cells (Park et al., 2013; Seo et al., 2012; Tse et al., 2013). In the current study, we demonstrate that decursin, a pyranocoumarin isolated from the Korean A.gigas, increased TRAIL-induced cell death in TRAIL-resistant NSCLC cells, in a synergistic manner. Targeting the main factors that induce TRAIL resistance is a common strategy to overcome resistance in cancers. 1040

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For example, inhibition of cell survival factors such as survivin, Bcl family members and Mcl-1, and/or anti-apoptotic factors, such as c-FLIP and IAPs, enhanced TRAIL sensitivity (Gillissen et al., 2010; Hellwig and Rehm, 2012; Johnson et al., 2003; Zang et al., 2014). In addition, induction of death receptors such as DR5 and DR4 was an effective method of restoring TRAIL sensitivity in cancer cells (Yamaguchi and Wang, 2004). A variety of natural compounds, including cryptotanshinone and 2-methoxyestradiol, induced DR5 and/or DR4 in TRAIL-resistant cancer cells, leading to increased apoptosis via TRAIL death signalling (LaVallee et al., 2003; Tse et al., 2013). Consistent with these reports, we demonstrate here that decursin also dramatically induces DR5, but not DR4, leading to sensitization to TRAIL-induced apoptosis in various NSCLC cell lines (Figure 3). Reactive oxygen species (ROS), which are chemically reactive molecules, are involved in cell signalling and homeostasis (Devasagayam et al., 2004). Also, ROS are generated in all cellular compartments during processes that involve consumption or production of oxygen, such as the mitochondrial respiratory chain and the ER (Stadtman, 2002). There is a close relationship between protein folding in the ER and generation of ROS. According to studies of ROS generation during ER stress, Ca2+ release from the ER lumen is a major

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Figure 6 Effect of ROS generation by decursin on ER stress induction and expression of ATF4/CHOP/DR5 in TRAIL-resistant NSCLC cells. (A) H1299 cells were treated with 60 μM decursin for the indicated times. (B) H1299 cells were treated with various concentrations of decursin (Dec) for 1 h, stained with 1 μM H2DCFDA for 40 min and then analysed by FACS. *** = P < 0.001 compared with the untreated control. FACS analysis was performed in triplicate, in three separate experiments. (C) Cells were treated with 60 μM decursin for 1 h, and then incubated with 1 μM H2DCFDA for 40 min. *** = P < 0.001 compared with control of each cell type. Data were obtained from three separate experiments. (D) A549 cells were pre-treated with 10 mM sodium pyruvate (SP) for 1 h and then treated with or without 60 μM decursin for 24 h. Cells were then lysed and subjected to Western blotting or (E) analysed for apoptosis by FACS. D + T; Decursin + TRAIL, D + T + SP; decursin+ TRAIL + sodium pyruvate. * = P < 0.01 and *** = P < 0.001 compared with the untreated control; # = P < 0.05 compared with the decursin and TRAIL-treated group. FACS analysis was performed in triplicate, in three separate experiments. (F) Inhibition of ROS generation by decursin in A549 cells stably transfected with catalase. A549 cells were transfected with human catalase/pCMV6A or empty vector and exposed to 60 μM decursin for 3 days. Then, catalase expression was confirmed in A549 control and catalase-transfected A549 cells by Western blotting. This experiment was repeated three times. (G) ROS production was measured in catalase-transfected A549 cells and untreated control by FACS analysis with 1 μM H2DCFDA staining for 40 min. FACS analysis was performed in triplicate, in three separate experiments. (H) Proposed scheme for apoptotic mechanism of decursin via selective ROS/ER/DR5 signalling axis.

stimulator of ROS production via increased mitochondrial Ca2+ loading (Berridge et al., 2003; Gorlach et al., 2006; Lizak et al., 2006). The increased ROS level in mitochondria further induces Ca2+ release from the ER via sensitization of Ca2+ channels in the ER membrane. Also, alterations in redox status or generation of ROS can directly or indirectly affect ER homeostasis and protein folding (Malhotra and Kaufman, 2007). Thus, in the current study, we focused on the roles of ER stress and ROS generation during decursin induced apoptosis in TRAIL-resistant NSCLC cells. Combined treatment with decursin and TRAIL generated production of ROS in a variety of lung cancer cell lines (Figure 6A–C), and induced activation of ER stress signalling, particularly through the PERK/ATF4/ CHOP pathway, while other ER stress signals were unaffected by the induction of ROS by decursin (Figure 5D and E). Stress of the ER stress induced expression of death receptors via ATF4/CHOP and/or ATF6/CHOP pathways (Averous et al., 2004; Gotoh et al., 2002) and particularly ER stress induced by oxidative agents regulated expression of survival or antiapoptotic proteins (Gillissen et al., 2010; Johnson et al., 2003). Consistent with these findings, we here have shown that ER stress induced by decursin markedly up-regulated

DR5 via induction of the PERK/ATF4/CHOP pathway of ER stress and down-regulated survivin and Bcl-xL in various TRAIL-resistant NSCLC cell lines (Figures 3–5, and Supporting Information Fig. S8). Nevertheless, the effects of ROS generation by decursin on other signalling pathways, such as toll-like receptor signalling (Woo et al., 2009), remains to be elucidated, hopefully in the near future. In summary, our findings have provided clear evidence that decursin induced DR5-mediated apoptosis, generated ROS production and selectively activated the PERK/ATF4/CHOP signalling in human lung cancer cells. Furthermore, decursin exerted synergistic effects with TRAIL to induce apoptosis. Thus this compound is a potent antitumor candidate, able to overcome chemoresistance of TRAIL-resistant NSCLC cells.

Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MEST) (no. 2012–0005755). British Journal of Pharmacology (2016) 173 1033–1044

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Author contributions J. K., E.-O. K., D.-B. J., G. W., J.H. J. and S.-H. K. performed the research. J. K. and M. Y. designed the research study. S.-H. K. supported grant for essential reagents and tools. J. K., B. K. and M. Y. analysed the data. M. Y. and S.-H. K. wrote the paper.

Conflict of interest The authors disclose no potential conflicts of interest.

Declaration of transparency and scientific rigour This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: http://dx.doi.org/10.1111/bph.13408 Figure S1 NSCLC cells exhibit variable sensitivity to TRAIL. TRAIL-induced cytotoxicity in NSCLC cells. Cells were exposed to TRAIL for 24 h, and cell viability was evaluated using an MTT assay. Figure S2 Decursin and TRAIL synergistically enhance cytotoxicity in NSCLC cell lines. Combination index (CI) values at the indicated Fa between Decursin and TRAIL in the A549, H596, Calu-1 and H1299 cell lines were calculated using the Calcusyn software. Figure S3 Synergistic cytotoxic effects of Decursin and TRAIL co-treatment on TRAIL-sensitive H460 cells. (A) H460 cells were co-treated with various concentrations of Decursin and 0, 20, 40 ng/ml TRAIL for 24 h. Cytotoxicity was assayed using an MTT assay. (B) Combination index (CI) values with fraction affected (Fa) between Decursin and TRAIL in H460 cells were calculated using the Calcusyn software. Figure S4 Effect of Decursin on XBP-1 splicing in H1299 cells. H1299 cells were treated with 60 uM Decursin for indicated time. Cells were lysed, total RNA was isolated and RT-PCR was performed to detect the spliced and unspliced forms of XBP-1. PCR products were separated on a 3% agarose gel. USF; Unspliced form, SF; Spliced form. Figure S5 Effect of Decursin-generated ROS on selective ER stress induction. H1299 cells were treated with (A) 60 μM Decursin for the indicated times, or (B) the indicated concentrations of Decursin for 60 min. Cells were stained with 1 μM H2DCFDA for 40 min, then analysed by FACS. Figure S6 Decursin does not induce ROS in normal lung cells. A normal lung cell line, Hel299, was treated with 60 μM Decursin for 1 h. Cells were stained with 1 μM H2DCFDA for 40 min, and then analysed by FACS. Figure S7. Attenuation of Decursin-mediated ROS induction by various ROS scavengers. A549 and H1299 were pre-treated British Journal of Pharmacology (2016) 173 1033–1044

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with each scavenger for 1 h and then treated with or without 60 μM Decursin for 3 h. Cells were then stained with antibodies for DR5, DR4 (green; FITC-labelled primary antibodies), or an IgG control (green; FITC-labelled antibody) and analysed for cell surface expression of DR4 and DR5 by flow cytometry. Figure S8 Downregulation of anti-apoptotic proteins by Decursin and/or TRAIL. A549 cells were treated with 30 μM Decursin(+), 10 ng/ml TRAIL(+), 60 μM Decursin(++) or 20 ng/ml TRAIL(++) alone or combination (Decursin(+)/TRAIL(+) or Decursin(++)/ TRAIL (++)) for 24 h. Whole-cell extracts were prepared and analysed by Western blotting using antibodies against Survivin, Bcl-xL, and Mcl-1; β-actin was used as the internal standard.

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Figure S9 Effect of DR5 antagonist on Decursin/TRAILinduced apoptosis. (A) A549, H1299 and H596 cells were treated with 60 μM Decursin and 20 ng/ml TRAIL with or without various concentrations of the DR5 antagonist rhTRAIL R2/FC chimera for 24 h. Cell viability was measured using an MTT assay. (B and C) H1299 cells were treated with 60 μM Decursin and 20 ng/ml TRAIL and/or 1 μg/ml rhTRAIL R2/FC chimera for 24 h. After staining with Annexin V-FITC and PI, apoptotic cells were analysed using a flow cytometer. *** = P < 0.01 compared to the untreated control; ## = P < 0.05 and ### = P < 0.01 compared to the Decursin and TRAIL treated group.

Decursin enhances TRAIL-induced apoptosis through oxidative stress mediated- endoplasmic reticulum stress signalling in non-small cell lung cancers.

The TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. Howe...
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