Morusin Induces TRAIL Sensitization by Regulating EGFR and DR5 in

Article pubs.acs.org/jnp

Morusin Induces TRAIL Sensitization by Regulating EGFR and DR5 in Human Glioblastoma Cells Dain Park,† In Jin Ha,⊥ Sang-Yoon Park,† Minji Choi,‡ Sung-Lyul Lim,† Sung-Hoon Kim,†,‡ Jun-Hee Lee,§,⊥ Kwang Seok Ahn,†,‡ Miyong Yun,*,‡,⊥ and Seok-Geun Lee*,†,‡,⊥ †

Cancer Preventive Material Development Research Center, ‡Department of Science in Korean Medicine, and §Department of Sasang Constitutional Medicine, College of Korean Medicine, Kyung Hee University, Seoul 02447, Republic of Korea ⊥ Korean Medicine Clinical Trial Center, Kyung Hee University Korean Medicine Hospital, Seoul 02447, Republic of Korea S Supporting Information *

ABSTRACT: Glioblastoma is one of the most malignant primary tumors, and the prognosis for glioblastoma patients remains poor. Tumor-necrosis-factor-related apoptosisinducing ligand (TRAIL) is considered a promising anticancer agent due to its remarkable ability to selectively kill tumor cells. However, since many cancers are resistant to TRAIL, strategies to overcome resistance are required for the successful use of TRAIL in the clinic. In the present study, the potential of morusin as a TRAIL sensitizer in human glioblastoma cells was evaluated. Treatment with TRAIL or morusin alone showed weak cytotoxicity in human glioblastoma cells. However, combination treatment of TRAIL with morusin synergistically decreased cell viability and increased apoptosis compared with single treatment. Morusin induced expression of death receptor 5 (DR5), but not DR4 or decoy receptors (DcR1 and DcR2). Furthermore, morusin significantly decreased anti-apoptotic molecules survivin and XIAP. In addition, morusin reduced expression of EGFR and PDFGR as well as phosphorylation of STAT3, possibly mediating down-regulation of survivin and XIAP. Together these results suggest that morusin enhances TRAIL sensitivity in human glioblastoma cells through regulating expression of DR5 and EGFR. Therefore, the combination treatment of TRAIL and morusin may be a new therapeutic strategy for malignant glioma patients.

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indicating that genetic alterations of EGFR are the major RTK lesions as well as the most common oncogenic alteration in glioblastoma.4 Another major subset of RTK in glioblastoma is platelet-derived growth factor receptor (PDGFR), amplification of which was detected in 13% of the patients, and nearly half of these tumors had amplifications and/or mutations in EGFR as well.4 EGFR and PDGFR induce their trans-phosphorylation after dimerization followed by activation of critical downstream signaling molecules such as AKT, STAT, and ERK, resulting in promotion of glioma growth and malignance.5,6 Especially, STAT3 controls a variety of death-related, anti-apoptotic, and pro-apoptotic proteins.7−10 Therefore, targeting both EGFR and PDGFR is regarded as a promising strategy to block glioblastoma proliferation and cell survival. Malignant tumors in general possess the ability to evade apoptosis and other death signals, which can result in drug resistance.11,12 For that reason, induction of cell death is regarded as a promising therapeutic strategy for cancer treatment. Caspase-dependent apoptosis is typically activated through either the intrinsic or extrinsic apoptotic pathways. The intrinsic apoptotic pathway is induced by endogenous stresses such as DNA damage, hypoxia, or other cellular stresses, but the extrinsic apoptotic pathway is mediated by cell death

lioblastoma is the second most frequently reported brain tumor type and the most common malignant tumor in the brain and central nervous system (CNS). Glioblastoma accounts for the majority of gliomas in the USA and has only a 5% five-year survival rate.1 In Korea, glioblastoma accounts for 15.1% of all primary brain and CNS tumors and is the most common neuroepithelial tumor, accounting for 34.4% of all gliomas.2 However, despite continuous efforts to develop effective molecular-targeted therapies, treatment for glioblastoma remains challenging due to several contributing factors including high rates of therapeutic resistance, redundancy of aberrantly activated signaling pathways, difficulty in effectively delivering therapeutics, and the remarkable spatial and temporal heterogeneity within individual tumors.3 Epidermal growth factor receptor (EGFR) is one of the receptor tyrosine kinases (RTKs) that are crucial regulators of cellular proliferation, metabolism, and survival in response to environmental cues.4 Therefore, it is not surprising that RTKs such as EGFR are overexpressed and mutated in various types of human cancer including glioblastoma, indicating their critical roles for tumor initiation and/or progression and resistance to cancer treatment.4 RTK amplifications and/or mutations occurred in 66% of primary glioblastoma patients tested by The Cancer Genome Atlas (TCGA), and amplifications and/or oncogenic mutations in EGFR were the only RTK lesions observed in 50% of all the primary glioblastoma patients, © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 23, 2015

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which TRAIL regulates cancer cell viability, we then investigated whether the cytotoxic effect of the combination treatment with TRAIL and morusin in glioblastoma cells is mediated by apoptosis. As shown in Figure 2A, the combination treatment of glioblastoma cells with TRAIL and morusin significantly increased protein levels of cleaved-caspase 3, 8, and 9 and cleaved-PARP, even though their concentrations were half that of the single treatment. Furthermore, increased cleavage of both caspase 8 and caspase 9 by the combination treatment indicated coincidental induction of both extrinsic and intrinsic apoptosis pathways. Consistently, flow cytometry analysis after annexin V−fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining revealed that the combination treatment of TRAIL and morusin synergistically increased the apoptotic population compared with single agent treatment in human glioblastoma cells (Figure 2B and C). Together these results suggest that morusin may be a novel potent TRAIL sensitizer in glioblastoma. Morusin Induces DR5 in Human Glioblastoma Cells. One of the crucial mechanisms for tumors to acquire TRAIL resistance is to down-regulate death receptors (DR4 and DR5) and/or up-regulate decoy receptors (DcR1 and DcR2).26−29 In order to elucidate how morusin overcomes TRAIL resistance in glioblastoma cells, expression levels of death receptors were evaluated after morusin treatment. As shown in Figure 3A and B, morusin strongly increased expression of DR5 in a concentration- and time-dependent manner, but had little effect on that of the other death receptors (DR4, DcR1, and DcR2) in glioblastoma cells. Consistent with these findings, morusin increased expression of DR5 but not DR4 on the cell membranes of glioblastoma cells (Figure 3C). In addition, RTqPCR analysis revealed that morusin induced mRNA expression of DR5 (Figure 3D). Furthermore, knockdown of DR5 decreased the combination treatment-mediated cell death about 66% (29% to 10% of cell death) (Figure 3E), indicating the crucial role of DR5 in the cytotoxic effect of the combination treatment of TRAIL with morusin in glioblastoma cells. Together these results suggest that morusin can act as a TRAIL sensitizer by up-regulating expression of DR5 in glioblastoma. Morusin Reduces XIAP and Survivin in Human Glioblastoma Cells. Another mechanism by which tumors can acquire TRAIL resistance is up-regulation of anti-apoptotic proteins.30,31 Therefore, we next examined whether morusin affects the expression of anti-apoptotic proteins in glioblastoma cells. As shown in Figure 4A and B, morusin decreased expression of XIAP and survivin, but not c-IAP1, in a concentration- and time-dependent manner in glioblastoma cells. In addition, morusin reduced expression of XIAP and survivin at the levels of mRNA in a concentration- and timedependent manner (Figure 4C and D). These results suggest that morusin increased TRAIL sensitivity in glioblastoma cells in part through reducing expression of the anti-apoptotic proteins XIAP and survivin. Morusin Inhibits the EGFR/PDGFR-STAT3 Signaling Pathway. RTKs such as EGFR and PDGFR play pivotal roles in glioma progression through boosting growth signals using master transcription regulators such as STAT3, target genes of which include anti-apoptotic genes.5,32 As shown in Figure 5A morusin significantly repressed expression of EGFR and PDGFR in glioblastoma cells. Morusin also decreased phosphorylation levels of EGFR (Figure 5B and C). In addition, morusin reduced levels of phosphorylated STAT3,

receptors such as the tumor necrosis factor (TNF) receptor superfamily.13−15 In humans, tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL), a TNF superfamily member, can interact 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) with a truncated cytoplasmic death domain.16,17 TRAIL-mediated apoptosis induction especially has been considered as an effective tumor therapy because of its ability to attack cancer cells selectively.18 However, various types of human cancers, including glioblastoma, have been known to be resistant to TRAIL-induced apoptosis, limiting its clinical application. Previous reports have proved that morusin isolated from the root bark of Morus alba L. (Moraceae) has various biological activities such as antimicrobial activity, scavenging activity against superoxide anion radical, and anti-inflammatory activity.19−21 Recent studies also reported that morusin can induce apoptosis via inhibition of NF-κB and STAT3 signaling pathways in human prostate cancer, hepatoma, and cervical cancer cells.22−24 The present study focused on the potential effect of morusin as a TRAIL sensitizer and the mechanism by which morusin induces TRAIL sensitization in human glioblastoma cells.

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RESULTS AND DISCUSSION Combination Treatment of TRAIL with Morusin Synergistically Increases Cytotoxicity in Human Glioblastoma Cells. In order to investigate the possible therapeutic effect of morusin and TRAIL in glioblastoma, we first examined whether a single treatment of morusin or TRAIL affects viability of human glioblastoma cells. Consistent with previous reports, 25 U87MG, U138MG, U373MG, and U251MG cells showed strong TRAIL resistance, with IC50 values of more than 400 ng/mL, while T98G and LN18 cells were sensitive to TRAIL, with IC50 values of less than 5 ng/mL (Figure 1A and Supporting Table 1). Before evaluation of anticancer effects of morusin in glioblastoma, we checked the stability of morusin in U87MG cell culture conditions. As shown in Supporting Figure 1, more than 94% of morusin remained in the cell culture media even after 24 h, indicating that morusin is very stable in the experimental conditions. As shown in Figure 1A and Supporting Table 1, morusin was cytotoxic in all the glioblastoma cell lines but lower in toxicity, with IC50 values of approximately 24−75 μM, with no significant cell death in normal astrocytes. Intriguingly, cotreatment with TRAIL and morusin was found to synergistically decrease the viability of glioblastoma cells (Figure 1B). Furthermore, the combination index (CI) values were less than 0.8 at almost all fraction-affected points (Figure 1C), implying strong synergy of TRAIL and morusin in induction of glioblastoma cell death. Combination Treatment of TRAIL with Morusin Synergistically Induces Apoptosis in Human Glioblastoma Cells. Since apoptosis is a canonical mechanism by B

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Figure 1. Effect of the combination treatment of TRAIL with morusin on cell viability in human glioblastoma cells. (A) Primary normal astrocytes and LN18, U87MG, U373MG, T98G, U251MG, and U138MG human glioblastoma cells were treated with TRAIL for 72 h or with morusin for 24 h, as indicated. (B) Glioblastoma cells were cotreated with TRAIL (100 ng/mL) and the indicated concentrations of morusin for 24 h. Cell viability was then analyzed using MTT assays. (C) Combination index values with fraction affected between TRAIL and morusin in glioblastoma cells were calculated using Calcusyn software.

of only the extrinsic apoptotic pathway by induction of death receptors is not enough. Therefore, induction of both the intrinsic and extrinsic apoptotic pathways would be a promising strategy in combination treatment using TRAIL against cancer. As shown in Figures 3 and 4, morusin simultaneously activates both the intrinsic (decrease of XIAP and survivin) and extrinsic (increase of death receptor DR5) apoptotic signals in glioblastoma cells, suggesting the combination treatment of TRAIL with morusin as a promising strategy for glioma therapy. Natural compounds used for combination therapy with TRAIL in general induce the death receptors, particularly DR5.43,44 Cryptotanshinone,42 2-methoxyestradiol,45 and decursin (unpublished data) increase DR5 through CCAAT/ enhancer binding protein homologous protein (CHOP), which is induced by reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Morusin-mediated production of ROS and ER stress may lead to the increase in DR5 (Figure 3) and CHOP (unpublished data). The JAK−STAT pathway has been demonstrated to be one of the most important signals for tumor progression. For that reason, the STAT pathway has been a common therapeutic target for anticancer agents.46 Recent studies reported that morusin inactivated STAT3 in prostate cancer and hepatoma. 22,23 The present study also proves that morusin significantly reduces STAT3 activity as well as its downstream targets XIAP and survivin in glioblastoma cells (Figure 4B and

which is a major downstream signaling molecule of the RTKs, while ERK activity was only marginally affected (Figure 5B and C). These results suggest that morusin may inhibit the EGFR/ PDGFR-STAT3 pathway though down-regulating expression of EGFR and PDGFR. Even though TRAIL is considered an important therapeutic agent for tumor treatment, clinical trials have revealed its limitations due to the acquisition of tumor resistance. There are lots of mechanisms by which tumor cells acquire TRAIL resistance, such as induction of decoy receptors (DcR1 and DcR2),29,33 anti-apoptotic proteins (c-FLIP, IAPs, Bcl2, Bcl-xL, and Mcl-1),34−36 and proliferation activators (PI3K, AKT, and NF-κB).37−39 Another major strategy to induce TRAIL resistance is to suppress death receptors DR5 and/or DR4.26,31 To establish effective therapeutic strategies for treating tumors resistant to TRAIL, much effort has focused on phytomedicine-based combination therapies. In particular, combining TRAIL with curcumin, kurarinone, or cryptotanshinone has been shown to increase the TRAIL sensitivity of several types of TRAIL-resistant cancers.40−42 In the present study, combination of TRAIL and morusin efficiently and synergistically increased cell death in human glioblastoma cells resistant to TRAIL, suggesting morusin may be a novel and potent TRAIL sensitizer (Figure 1C and D). To increase the sensitivity of cancer cells to TRAIL, a variety of approaches have been attempted such as up-regulation of death receptors DR4 and/or DR5.26,31 However, amplification C

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Figure 2. Effect of the combination treatment of TRAIL with morusin on apoptosis induction in human glioblastoma cells. (A) U87MG and T98G cells were treated with TRAIL (50 ng/mL) or morusin (5 μM) alone or with TRAIL (25 ng/mL) and morusin (2.5 μM) in combination for 24 h. Cell lysates were prepared and subjected to Western blotting with the indicated antibodies. β-Actin was used as an internal control. (B) Cells were treated with TRAIL (50 ng/mL) and morusin (20 μM) for 24 h as indicated. The cells were then stained with annexin V-FITC and PI and analyzed using flow cytometry. (C) Percentage of apoptotic cells (annexin V-FITC positive) was shown by a histogram. Data in the graphs are presented as the mean ± SD (**, p < 0.01 and ***, p < 0.001 versus mock control).

Figure 3. Effect of morusin on expression of death receptors and decoy receptors in human glioblastoma cells. (A) U87MG, U373MG, U251MG, and LN18 cells were treated with morusin for 24 h as indicated. (B) U87MG and U373MG cells were treated with morusin (25 μM) as indicated. Cell extracts were prepared and subjected to Western blotting with the indicated antibodies. β-Actin was used as an internal control. (C) U87MG, U373MG, and U251MG cells were treated with 25 μM morusin for 24 h. Cells were then stained with FITC-conjugated antibodies for DR4, DR5, or IgG control, and expression of DR4 and DR5 on the cell surface was analyzed using flow cytometry. (D) U87MG cells were treated with 25 μM morusin as indicated. Total RNA was isolated from the treated cells, and expression of DR5 mRNA was determined by RT-qPCR. (E) U87MG cells were transiently transfected with control siRNA or DR5 siRNA. Cells were cotreated with TRAIL (50 ng/mL) and morusin (20 μM) 48 h after transfection for 24 h as indicated. Cell viability was then measured using MTT assays. Data in the graphs are presented as the mean ± SD (*, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus mock control).

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Figure 4. Effect of morusin on expression of anti-apoptotic proteins in human glioblastoma cells. (A) U87MG and U373MG cells were treated with morusin for 24 h as indicated. (B) U87MG and U373MG cells were treated with 25 μM morusin as indicated. Whole cell extracts were then prepared and analyzed by Western blotting with the indicated antibodies. β-Actin was used as an internal control. (C) U87MG cells were treated with morusin for 24 h as indicated. (D) U87MG cells were treated with 25 μM morusin as indicated. Total RNA was isolated from the treated cells, and mRNA expression of XIAP (■) and survivin (□) was determined by RT-qPCR. Data are presented as the mean ± SD (*, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus mock control).

PDGFR, Notch, and IL receptors.5,6,32,46 Recent reports have indicated that EGFR and PDGFR play a crucial role in glioma progression and drug resistance through activating STAT3.32,47,48 Thus, many anticancer drugs targeting mutant EGFR such as gefitinib, erlotinib, and BIBW2992 have been developed and used clinically.49,50 Despite clinical efficacy, issues remain to be resolved such as recurrence and resistance due to activation of various other growth factor receptors and their downstream signaling pathways.51,52 Therefore, targeting multiple pathways may be beneficial to cancer treatment. Inhibition of multiple RTKs by 17-allyloamino-17-demethoxygeldanamycin (17-AAG) showed greater effect on induction of cancer cell death than inhibition of individual RTKs.53 Morusin also substantially repressed expression of both EGFR and PDGFR in glioblastoma cells (Figure 4A), indicating its inhibition of multiple RTKs and suggesting it as a potentially good drug candidate for treating glioma. However, specific mechanisms by which morusin reduced expression of the RTKs have yet to be elucidated. In conclusion, this study demonstrates that morusin can sensitize glioblastoma cells to TRAIL-induced cell death. Morusin increased the death receptor DR5 and suppressed the STAT3 pathway via down-regulating EGFR and PDGFR, resulting in sensitizing glioblastoma cells to TRAIL. Taken together, these results suggest that morusin could be a novel potent TRAIL sensitizer for glioblastoma, and therefore the combination treatment of TRAIL with morusin would be a potentially good therapeutic strategy for glioblastoma patients.

Figure 5. Effect of morusin on the EGFR pathway in human glioblastoma cells. (A) LN18, U87MG, T98G, U251MG, and U373MG glioblastoma cells were treated with morusin for 24 h. (B) U87MG and U373MG cells were treated with morusin for 24 h as indicated. (C) U87MG and U373MG cells were treated with 25 μM morusin as indicated. Whole cell lysates were then prepared and analyzed by Western blotting with the indicated antibodies. β-Actin was used as an internal control.

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EXPERIMENTAL SECTION

Cell Cultures and Reagents. Human glioblastoma cell lines U251MG and LN18 were previously described.54,55 U87MG, T98G, and U373MG were purchased from the KCLB (Korean Cell Line Bank). U138MG was a kind gift from Dr. Sin-Soo Jeon (Catholic University of Korea College of Medicine, Seoul, Republic of Korea). U251MG and U138MG were cultured in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic. U87MG and T98G were cultured in MEM, and U373MG was cultured in RPMI supplemented with 10% FBS and 1% antibiotic/antimycotic. Primary normal

C), which suggests that STAT3 may be a target of morusin associated with glioblastoma cell death. STAT3 activation is mediated by various receptor proteins including EGFR, E

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were analyzed by FACS Calibur flow cytometry (BD Biosciences, San Jose, CA, USA). Apoptosis Assay. Cells ((2−3) × 105 cells/well) in six-well plates were treated with the indicated compounds for 24 h and stained with an annexin V-FITC and PI kit (Bio Vision Technology Inc., Golden, CO, USA). Then the cells were analyzed using FACS Calibur flow cytometry. Combination Index Calculation between Morusin and TRAIL. To determine the synergy between TRAIL and morusin, cytotoxicity assays were performed in the cells treated with 100 ng/mL of TRAIL and morusin, the concentrations of which were gradually increased. The fraction of living cells at each concentration was used for the analysis of synergism between TRAIL and morusin using the CalcuSyn software (Biosoft, MO, USA). Statistical Analysis. Data are presented as mean ± standard deviation (SD) from at least three independent experiments in triplicate or more and analyzed for statistical significance using the unpaired Student’s t-test. p < 0.05 was considered significant.

astrocytes were isolated from the cortex of P1 to P4 ICR (Institute of Cancer Research) mice (DBL Co., Ltd., Eumseong, Chuncheongbukdo, Republic of Korea) and maintained in astrocyte culture media (high-glucose DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin) as described.56 All applicable international and institutional guidelines for the care and use of animals were followed, and the study using the primary brain cells isolated from the mice has been approved by the research ethics committee at Kyung Hee University (KHUASP(SE)-14-056). All cells were cultured in a humidified incubator with 5% CO2 at 37 °C, and the viability of cultured cells was monitored by a LUNA-FL automated cell counter (Logos Biosystems, Anyang, Gyeonggi-do, Republic of Korea). Recombinant human TRAIL/Apo2L was purchased from ATGen (Seongnam, Gyeonggi-do, Republic of Korea). Morusin (BP0961, purity (HPLC, 270 nm) ≥98%) purchased from Biopurify Phytochemicals Ltd. (Chengdu, Sichuan, People’s Republic of China) was dissolved in DMSO. Cell Viability Assay. Cell viability was measured by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cells ((3−5) × 103 cells/well) were seeded in 96-well plates, and 1 day after seeding the cells were treated with various concentrations of morusin and TRAIL. After the indicated time, the medium was removed, and fresh medium containing 1 mg/mL MTT was added to each well. The cells were incubated at 37 °C for 2 h, and then an equal volume of MTT lysis buffer was added to each well. The cells were incubated at 37 °C overnight. OD at 570 nm was measured using a microplate reader (Tecan Austria GmbH, Grödig, Austria). Western Blotting Analysis. Whole cell lysates were prepared, and Western blotting was performed as described.23 Primary antibodies against cleaved-PARP, cleaved-caspase 3, cleaved-caspase 8, cleavedcaspase 9, XIAP, ERK, phospho-ERK, phospho-EGFR, EGFR, PDGFR, STAT3, and phospho-STAT3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against DR4, DR5, cIAP1, and survivin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against β-actin, DcR1, and DcR2 were purchased from Sigma-Aldrich. Secondary antibodies HRP-conjugated anti-mouse IgG and anti-rabbit IgG (1:5000; Cell Signaling Biotechnology) were used for immunoblotting. Transient Transfection of DR5 siRNA. Cells were transiently transfected with a validated scrambled control siRNA or specific siRNA (Mbiotech, Hanam, Gyeonggi-do, Republic of Korea) for DR5 using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) in six-well plates. Sequences for DR5 siRNA: sense, AAUGAGAUAAAGGUGGCUAAAGCTG, and antisense, CAGCUUUAGCCACCUUUAUCUCAUUGU. Two days after transfection the cells were treated with TRAIL and morusin. Real-Time Quantitative PCR (RT-qPCR). Total RNA was isolated using TRI Reagent solution (Ambion, Waltham, MA, USA) according to the manufacturer’s instructions. Total RNA (1 μg) isolated from cells was reverse transcribed to cDNA using PrimeScript first-strand cDNA synthesis kit (Takara Korea Biomedical Inc., Seoul, Republic of Korea) according to the manufacturer’s instructions. Amplification of each cDNA was monitored using Sensi FAST SYBR No-ROX kit (Bioline, Taunton, MA, USA) on a LightCycler instrument (Roche Applied Sciences, Indianapolis, IN, USA) according to the manufacturer’s protocol. RT-qPCR used the following specific primers: DR5 (forward: 5′-GACTCTGAGACAGTGCTTCGATGA-3′, and reverse: 5′-CCATGAGGCCCAACTTCCT-3′), XIAP (forward: 5′-GCAAGAGCTCAAGGAGACCA-3′, and reverse: 5′-AAGGGTATTAGGATGGGAGTTCA-3′), Survivin (forward: 5′TTGGCCCAGTGTTTCTTCTGCTTC-3′, and reverse: 5′GCACTTTCTCCGCAGTTTCCTCAA-3′). GAPDH was used as an internal control. Expression of Death Receptors on the Cell Surface. To analyze expression of DR4 and DR5 on the cell surface, cells were incubated with 0.5 μg/mL of FITC-conjugated DR4, DR5, or isotype control IgG antibody (Abcam, Cambridge, MA, USA) in FACS buffer for 30 min on ice. After washing with FACS buffer three times, cells

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00919. Additional information (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +82-2-958-9590. E-mail: [email protected]. *Tel/Fax: +82-2-961-2355. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Sin-Soo Jeon (Catholic University of Korea College of Medicine, Seoul, Republic of Korea) for the cell line U138MG. This work was supported by research grants (20070054391, NRF-2013R1A2A2A01069099, and NRF2014R1A1A2056230) from the National Research Foundation of Korea and a grant (grant number 1320120) from the National R&D Program for Cancer Control of Ministry of Health and Welfare in Republic of Korea.

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DOI: 10.1021/acs.jnatprod.5b00919 J. Nat. Prod. XXXX, XXX, XXX−XXX