Mol Cell Biochem (2011) 350:169–175 DOI 10.1007/s11010-010-0695-z

Gomisin N enhances TNF-a-induced apoptosis via inhibition of the NF-jB and EGFR survival pathways Pornthip Waiwut • Myoung-Sook Shin • Akiko Inujima • Yue Zhou • Keiichi Koizumi Ikuo Saiki • Hiroaki Sakurai

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Received: 10 August 2010 / Accepted: 10 December 2010 / Published online: 28 December 2010 Ó Springer Science+Business Media, LLC. 2010

Abstract Tumor necrosis factor (TNF-a) is a pleiotropic cytokine that plays an important role in the control of cell proliferation, differentiation, and apoptosis. TNF-ainduced apoptosis is limited by TAK1-mediated activation of NF-jB (mainly p65-p50 hetrodimer) signaling pathway. We have recently reported that TAK1 regulates phosphorylation of EGFR at Ser-1046/7 through p38 MAPK, which cooperates with NF-jB in TNF-a-induced apoptosis. The present study investigated the effect of gomisins A and N, dibenzocyclooctadiene lignans isolated from the fruit of Schisandra chinensis, on TNF-a-induced apoptosis in HeLa cells. Gomisins A and N strongly promoted TNF-ainduced cleavage of caspase-3 and PARP-1, which are key markers of apoptosis. We found that gomisin N, but not gomisin A, inhibited the TNF-a-induced activation of NFjB by suppressing the activation of IKKa. Gomisin N also inhibited p38-mediated phosphorylation of the EGFR at Ser-1046/7 and subsequent endocytosis of EGFR, another prosurvival pathway. The findings suggested that gomisin N enhanced TNF-a-induced apoptosis by suppressing of NF-jB and EGFR signaling pathways. Keywords TNF-a
NF-jB
EGFR
Apoptosis
Gomisin
Schisandra chinensis

P. Waiwut
M.-S. Shin
A. Inujima
Y. Zhou
K. Koizumi
I. Saiki
H. Sakurai (&) Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan e-mail: [email protected]

Introduction The fruit of Schisandra chinensis has been used in traditional Chinese medicine to treat a variety of diseases such as diabetes, asthma, respiratory infectious, and mental illness such as palpitation, insomnia, and forgetfulness. It is used to tone up and strengthen kidney function and to help the body to balance levels of fluid, making it helpful for treating excessive sweating. S. chinensis has also been employed in the treatment and prevention of some chronic diseases such as inflammation, hepatitis, and cancer. The major bioactive constituents of S. chinensis are lignans, including gomisins A, B, C, E, F, G, K, N, and J, scisandrol B, and schisandrin C. [1, 2]. Pharmacological studies revealed that lignans isolated from S. chinensis show anti-cancer, anti-hepatotoxic, anti-oxidative, and anti-inflammatory activities [3–5]. Gomisins A and N are dibenzo[a,c]cyclooctadiene lignans (Fig. 1a) with the R- and S-biphenyl configurations, respectively [6–8]. Gomisin A shows anti-apoptotic activity and protects the liver from hepatotoxic chemicals [9]. In contrast, gomisin N induces apoptosis of human hepatic carcinoma cells [10]. However, little is known about molecular mechanisms for the apoptosisinducing activity of gomisins A and N in human cancer cells. Tumor necrosis factor alpha (TNF-a) stimulates several intracellular signaling pathways, including nuclear factor-jB (NF-jB) and mitogen-activated protein kinases (MAPKs) [11]. TNF-a also triggers apoptosis via formation of the deathinducing signaling complex (DISC) [11]. This complex consists of trimerized receptors, the death domain-containing adaptor protein FADD (Fas-associated death domain protein) and caspase-8. Activation of caspase-8 leads to the direct activation of downstream caspases, such as caspase-3, and subsequently the cleavage of a nuclear enzyme, poly (ADPribose) polymerase (PARP). Transforming growth factor-bactivated kinase (TAK1) is a common upstream regulator of

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Fig. 1 Effects of gomisins A and N on TNF-a-induced cell death. a Chemical structure of gomisins A and N. b HeLa cells were treated with gomisins A and N in the presence or absence of TNF-a (20 ng/ml) for 24 h. Cell viability was determined by WST-1 assay. *P \ 0.05

NF-jB and MAPKs and prevents the apoptotic responses. The IjB kinase (IKK) complex phosphorylates NF-jB at Ser-536 as well as IjBa at Ser-32/36 in cytokine-induced NF-jB activation [12–14]. NF-jB is involved in cell survival in a variety of cell types by up-regulating the expression of antiapoptotic genes, including c-FLIP and A20 [15]. We have recently demonstrated that TNF-a triggers a non-canonical epidermal growth factor receptor (EGFR) pathway in a TAK1-dependent fashion, in which EGFR Ser1046/7 and Thr669 are phosphorylated via an extracellular signal-regulated kinase (ERK) and p38 MAPK, respectively. Moreover, p38-mediated Ser phosphorylation triggers transient endocytosis of the EGFR, which blocks the TNF-a death signal [16, 17]. Therefore, TAK1 regulates two independent survival pathways, the NF-jB and EGFR pathways, to prevent TNF-ainduced cell death. The present study investigated the effect of gomisins A and N on TNF-a signaling in HeLa cells. We found that gomisin N, but not gomisin A, inhibited the TNF-a-induced NF-jB and EGFR signaling pathways, which resulted in enhanced pro-apoptotic events.

Materials and methods Antibodies and reagents The anti-phospho p65 (Ser-536), IKKa (Ser-176/180), IjBa (Ser-32/36), ERK (Thr-202, Tyr-204), MKK3/6 (Ser-189/

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207), p38 (Thr-180/Tyr-182), and EGFR (Thr-669 and Ser1046/1047), PARP-1 and caspase-3 antibodies were purchased from Cell Signaling Technology. The anti-phospho TAK1 antibody was generated as described previously [18]. Antibodies against TAK1 (M579), p38 (C-20-G), JNK, ERK1 (C-16), p65 (C-20-G), IjBa (C-21), EGFR (1005), Lamin B (C-20), and Actin (C-11) were obtained from Santa Cruz Biotechnologies. Recombinant human TNF-a was obtained from R&D System. Gomisins A and N were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cycloheximide was purchased from Sigma. Cell culture and cytotoxicity assay HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 lg/ml streptomycin at 37°C in 5% CO2. The quantification of cell viability was performed using the cell proliferation reagent WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H5-tetrazolio]-1,3-benzene disulfonate) (DOJINDO, Kumamoto, Japan). HeLa cells were plated in 96-well microplates at 6 9 103 cells/wells and then incubated for 24 h. Gomisin-containing medium was added to the wells, and cells were incubated for 30 min and then stimulated with TNF-a. After a 24-h incubation, 10 ll of WST-1 solution was added, and the absorbance at 450 nm was measured. Preparation of cell extracts Cells were treated with gomisins A, N, and TNF-a, and whole cell lysates were prepared with lysis buffer [25 mM HEPES pH 7.7, 0.3 mM MgCl2, 0.2 mM EDTA, 10% Triton X-100, 20 mM b-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 10 lg/ml aprotinin, and 10 lg/ml leupeptin]. Cell lysate was collected from supernatant after centrifugation at 14,000 rpm for 10 min. Nuclear extracts were prepared as described previously [12]. In brief, cells were suspended in 420 ll of buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, 20 mM b-glycerophosphate, 0.1 mM sodium orthovanadate, 10 lg/ml aprotinin and 10 lg/ml leupeptin] and chilled on ice for 15 min. Next, 25 ll of 10% Nonidet P-40 was added, and the suspension was vigorously vortexed for 10 s. The nuclear pellets were suspended in 50 ll of buffer B [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 20 mM b-glycerophosphate, 1 mM sodium orthovanadate, 10 lg/ml aprotinin and 10 lg/ml leupeptin]. The mixture was kept on ice for 15 min with frequent agitation. Nuclear extracts were prepared by centrifugation at 15,000 rpm for 5 min.

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Immunoblotting Cell lysate was resolved by SDS-PAGE and transferred to an Immobilon-P-nylon membrane (Millipore). The membrane was treated with BlockAce (Dainippon Pharmaceutical Co. Ltd, Suita, Japan) and probed with primary antibodies. The antibodies were detected using horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and antigoat IgG (DAKO, Glostrup, Denmark) and visualized with the enhanced chemiluminescence system (Amersham Biosciences). Luciferase assay HeLa cells were transfected with a luciferase reporter plasmid under the control of 49 jB sites containing a neo resistance gene. A stable clone (HeLa-jB6) was isolated in medium containing 500 lg/ml G418. Cells (5 9 104) were seeded in a 96-well plate and stimulated with TNF-a for 6 h. The luciferase activity was measured by using the Dual-Luciferase reporter assay system (Promega).

FACS analysis HeLa cells were harvested in PBS, fixed with 2% paraformaldehyde for 20 min at room temperature, resuspended in 100 ll of FACS buffer (PBS containing 0.5% BSA and 0.05% NaN3) containing 1 lg of anti-EGFR monoclonal antibody (clone LA1; Upstate) and incubated on ice for 30 min. After being washed with FACS buffer, cells were incubated with FITC-conjugated anti-mouse IgG antibody (DAKO) on ice for 30 min and analyzed with the FACSCalibur system (BD).

Results Gomisin N enhances TNF-a-induced apoptotic responses Gomisin has been shown to induce apoptotic responses in cancer cells. We first confirmed the effects of gomisins A and N on TNF-a-induced apoptosis in HeLa cells. In the absence of TNF-a, both gomisins A and N showed cytotoxicity in a concentration-dependent manner (Fig. 1b). Interestingly, gomisin N at 100 lM, but not gomisin A, significantly enhanced TNF-a-induced cells death (Fig. 1b). As shown in Fig. 2, TNF-a induced cleavage of caspase-3 and PARP, cellular pro-apoptotic responses, although caspase-3 activation was very weak. Although both gomisins A and N marginally induced the apoptotic responses, they strongly enhanced TNF-a-induced cellular

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Fig. 2 Effects of gomisins A and N on TNF-a-induced apoptotic responses. HeLa cells were pretreated with gomisins A, N (100 lM) and cycloheximide (CHX, 25 lg/ml) for 30 min, and then stimulated with TNF-a (20 ng/ml) for 6 h. Whole cell extract was prepared, fractionated, and analyzed by Western blotting using anti-caspase-3, PARP, and actin antibodies. The arrows indicate cleaved forms of caspase-3 and PARP

pro-apoptotic responses in a similar extent by cycloheximide (Fig. 2). The apoptosis-promoting activity of gomisin N was stronger than that of gomisin A. These results indicate that gomisins A and N enhance TNF-a-induced apoptosis. Effect of gomisins A and N on TNF-a-induced p65 activation The effect of gomisins A and N on the TNF-a-induced activation of NF-jB was examined in HeLa cells stably transfected with an NF-jB-dependent reporter plasmid. Cells were pretreated with gomisins A and N for 30 min and then stimulated with TNF-a for 6 h. We found that gomisin N, but not gomisin A, inhibited TNF-a-induced NF-jB activation (Fig. 3a). We next performed immunoblotting to examine the activation of the TNF-a-triggered NF-jB pathway. Figure 3b and c shows that gomisin N inhibited the phosphorylation of IKKa and p65 as well as the phosphorylation and degradation of IjBa in a concentration-dependent manner. In addition, nuclear localization of p65 was inhibited by gomisin N (Fig. 3d). In contrast, the phosphorylation of TAK1 was not affected (Fig. 3b), indicating that gomisin N acted downstream of TAK1. Similar to the reporter gene assay, gomisin A had no effect on the TNF-a-induced phosphorylation of TAK1, IKKa, or p65 (Fig. 3b). These experiments indicate that gomisin N is able to inhibit the TNF-a-induced canonical NF-jB signaling pathway at the level of IKKa activation. Effect of gomisin on TNF-a-induced EGFR activation Apart from the TNF-a-induced activation of p65, we investigated the effects of gomisin on the TAK1-mediated

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Fig. 3 Effect of Gomisin N on the TNF-a-induced activation of p65 signaling. a HeLa cells transfected with NF-jB luciferase reporter plasmids were treated with DMSO, gomisins A and N at 100 lM for 30 min and stimulated with 20 ng/ml TNF-a for 4 h. *P \ 0.05. b HeLa cells were left untreated (lane 1), or pretreated with DMSO (lane 2), gomisin A (lane 3), or gomisin N (lane 4) at 100 lM. c The cells were pretreated with various concentrations of gomisin N (10, 30, and 100 lM) for 30 min, and then stimulated with TNF-a 20 ng/ ml for 5 min. Whole cell extract was prepared, fractionated, and analyzed by Western blotting using antibodies against phospo-TAK1, phospho-IKKa, phospho-IjBa, phospho-p65, TAK1, IKKa, IjBa, and p65. d After treatment with gomisin N (100 lM) and TNF-a, nuclear extracts were immunoblotted with anti-p65 and anti-Lamin B antibodies

phosphorylation of the EGFR at Ser-1046/7 and Thr-669, another anti-apoptotic pathway in TNF-a signaling. Figure 4a shows that gomisin N, but not gomisin A, inhibited TNF-a-induced Ser-1046/7 phosphorylation, whereas Thr-669 phosphorylation was not affected. We have reported that phosphorylation of the EGFR at the Ser and Thr residues is mediated via p38 and ERK pathways, respectively. This was correlated with the observation that gomisin N inhibited the activation p38, but not ERK (Fig. 4a), indicating selective inhibition of the p38-EGFR (Ser-1046/7) pathway. In addition, a similar concentration dependency was observed in the inhibition of IKKa and EGFR phosphorylation by gomisin N (Fig. 4b). Moreover, we have shown that p38-mediated Ser-1046/7 phosphorylation triggers TNF-a-induced endocytosis of the EGFR. A FACS analysis of the cell surface expression of the EGFR confirmed that SB203580, a p38 inhibitor, significantly inhibited the receptor’s internalization. Figure 4c shows that the TNF-a-induced internalization of the EGFR, in parallel with the Ser-1046/7 phosphorylation, was partially inhibited by gomisin N.

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We have previously demonstrated that p38 is a kinase downstream of TAK1 in HeLa cells. Since gomisin N did not block TAK1 phosphorylation, it is possible that MKK3/ 6, MAP2Ks connecting TAK1 and p38, are a target for gomisin N. To address this possibility, we further characterized activation of the TAK1-p38 pathway using chemical inhibitors of TAK1 and p38. The TAK1 inhibitor 5Z-7-oxozeaenol suppressed the TNF-a-induced autophosphorylation of TAK1, which resulted in the decrease in phosphorylation of its downstream kinases, including MKK3/6, p38, and MK2 (Fig. 5). In contrast, SB203580 inhibited only a downstream kinase, MK2 (Fig. 5). It has been shown that the p38 kinase activity controls feedback inhibition of TAK1 via phosphorylation of its adaptor protein, TAB 1 [17, 18]. Therefore, SB203580 abrogated the negative feedback regulation, which resulted in enhanced phosphorylation of TAK1. Gomisin N partially inhibited TNF-a-induced phosphorylation of MKK3/6, p38, and MK2; however, it did not block phosphorylation of TAK1 (Fig. 5). Collectively, these results suggested that gomisin N did not inhibit TAK1 kinase activity directly.

Discussion Gomisin N, a dibenzocyclooctadiene lignan isolated from the fruit of S. chinensis, has been reported as an anti-cancer drug candidate. Recent study demonstrated that gomisin N inhibits proliferation and induces apoptosis in human hepatic carcinomas through a mitochondrial (intrinsic) pathway by enhancing Bax protein expression and so in inhibiting the anti-apoptotic activity of Bcl-2 [10]. Gomisin A, with a similar molecular formula to gomisin N, also exhibited apoptosis-inducing activity in cancer cell lines [9, 19]. However, the mechanisms and signaling pathways for these compounds have not been clarified. Apoptosis can be initiated in two ways, via an extrinsic (death receptor mediated) pathway and intrinsic pathway (mitochondrialmediated) [20, 21]. Hence, in the present study, the effects of gomisins A and N on the extrinsic pathway were investigated by focusing on two independent TNF-ainduced and TAK1-regulated prosurvival signaling pathways, the NF-jB and MAPK-EGFR pathways. We confirmed that both gomisins A and N enhanced TNF-a-induced apoptosis by activating pro-apoptotic cleavage of caspase-3 and PARP-1 in HeLa cells. Gomisin N showed inhibitory effects on both NF-jB and the EGFR in TNF-a-triggered pathways downstream of the TAK1 kinase. However, gomisin A had no inhibitory effect on these pathways. Although the orientation, configuration,

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Fig. 4 Effect of gomisin N on TNF-a-induced EGFR signaling. a HeLa cells were left untreated (lane 1), or pretreated with DMSO (lane 2), gomisin A (lane 3), or gomisin N (lane 4) at 100 lM. b Cells were pretreated with various concentrations of gomisin N (10, 30, and 100 lM) for 30 min, and then stimulated with TNF-a 20 ng/ml for 10 min. Whole cell extract was prepared, fractionated, and analyzed by Western blotting using antibodies against phospho-EGFR (phospho-T699 and phospho-S1046/7), phospho-ERK, phospho-p38, EGFR, ERK, and p38. c Cells were pretreated with SB203580 (5 lM) or gomisin N (100 lM) for 30 min and then stimulated with TNF-a 20 ng/ml for 15 min. Cell surface expression of the EGFR was investigated by FACS analysis

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and chirality of their structures would influence the activity, it is necessary to investigate the molecular mechanisms responsible for the pro-apoptotic potential of gomisin A. In the TNF-a-induced NF-jB pathway, gomisin N inhibited the phosphorylation of IKKa and p65 but not TAK1, suggesting that gomisin N interferes with IKKa and multiple upstream kinases including NIK, MEKK3, Tpl2, PKC, and Akt [22]. In the TNF-a-induced EGFR signaling pathway, treatment cells with gomisin N resulted in inhibition of the phosphorylation of MKK3/6, p38, EGFR (pS1046/7), and MK2 but not TAK1. These results raise the possibility that gomisin N blocks the activation of MKK3/6 by interfering with some regulatory kinases including MEKK1-4, MLK, ASK1, and TAO, which have been shown to act as upstream kinases regulating MKK3/6 phosphorylation [23]. Identification of the target molecules is essential and will contribute to our understanding of the signaling complex on TAK1. The caspase-3 family plays an important role in driving apoptosis via both the extrinsic and intrinsic pathways [24]. We found treatment with only gomisins A or N without TNFa-stimulation induced cleavage of caspase-3 and PARP-1. The result suggested that these compounds might induce apoptosis through activation of the mitochondrial-mediated

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Fig. 6 Schematic diagram of the anti-apoptotic activity of gomisin N. Gomisin N enhances TNF-a-induced apoptosis via inhibition of the p65 and EGFR signaling pathways

intrinsic caspase-3 pathway. Additionally, gomisin A enhanced TNF-a-induced pro-apoptotic responses, cleavage of caspase-3 and PARP-1, without affecting TNF-ainduced NF-jB and EGFR activation. Gomisin N might therefore enhance TNF-a-induced apoptosis through another pathway such as the JNK pathway [25]. In summary, we showed that gomisin N enhanced TNFa-induced apoptosis by targeting proteins downstream of TAK1. Our findings indicated that gomisin N acted to suppress the activation of p65 through inhibition of IKKa phosphorylation and to suppress the p38-EGFR pathway through inhibition of MKK3/6 (Fig. 6). These results provide a new molecular basis for the development of gomisin N as a potential anti-cancer drug. Acknowledgments This work was supported in part by Grants-inAid for Challenging Exploratory Research (No. 09002374) and for the Knowledge Custer Initiative Toyama/Ishikawa Region (Hokuriku Innovation Cluster for Health Science) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and a grant from the First Bank of Toyama Foundation.

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