Prenylated Flavonoids and Resveratrol Derivatives Isolated from

Dec 24, 2014 - Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand. •S Supporting Information. ABSTRACT: In a screening program ...
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Prenylated Flavonoids and Resveratrol Derivatives Isolated from Artocarpus communis with the Ability to Overcome TRAIL Resistance Kazufumi Toume,† Tadashi Habu,† Midori A. Arai,† Takashi Koyano,‡ Thaworn Kowithayakorn,§ and Masami Ishibashi*,† †

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Temko Corporation, 4-27-4 Honcho, Nakano, Tokyo 164-0012, Japan § Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand ‡

S Supporting Information *

ABSTRACT: In a screening program on natural products that can abrogate tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance, four new prenylated flavonoid and resveratrol derivatives (1−4) were isolated from Artocarpus communis, together with eight known prenylflavonoids (5−12). The structures of 1−4 were elucidated spectroscopically. Pannokin E (1) (2 μM) and artonin E (5) (3 μM) potently exhibited the ability to overcome TRAIL resistance. Artonin E (5) induced caspase-dependent apoptosis in combination with TRAIL, increased caspase 3/7 activity, and enhanced the protein levels of p53 and DR5. Moreover, this substance decreased cell viability in combination with TRAIL and enhanced the protein levels of DR5, and these effects were mediated by increases in the production of ROS (reactive oxygen species). Thus, artonin E (5) was found to induce extrinsic apoptotic cell death by the ROSand p53-mediated up-regulation of DR5 expression in AGS cells.

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the CHOP-dependent up-regulation of DR5 expression, resulting in apoptosis in AGS cells.9 In the present investigation, the MeOH extract of Artocarpus communis Forst. (Moraceae) roots was found to reduce TRAIL resistance in a preliminary screening study. In order to identify additional bioactive natural products with the ability to overcome TRAIL resistance, A. communis was selected for further work. A. communis is a species common to Southeast Asia and is known as “bread fruit”. Several prenylated flavonoids have been isolated from the heartwood of this plant.10 Described herein are the activity-guided isolation and structure elucidation of four new prenylated flavonoids and resveratrol derivatives, named pannokins D−G (1−4), which were obtained along with eight known compounds (5−12) from A. communis roots. These isolated compounds have been investigated for their effects on TRAIL resistance.

umor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) may be useful for potential anticancer therapies, because it can induce tumor-cell-selective damage. TRAIL binds to death receptors such as death receptor 5 (DR5) and/or DR4 and induces the formation of the death-inducing signaling complex (DISC), which activates caspase-signaling pathways, leading to apoptosis (an extrinsic or death-receptor pathway). In other cells or conditions, activation of the intrinsic (mitochondrial) pathway is known to be required for apoptosis.1,2 However, many cancer cells, particularly those in highly malignant tumors, have been revealed to exhibit resistance to TRAIL. Therefore, small molecules with the ability to overcome TRAIL resistance are expected to constitute an important strategy for the development of anticancer agents. Previously, bioactive natural products have been screened that can cause tumor-selective apoptosis-inducing activity,3,4 and the MeOH-soluble extracts of several medicinal plants collected in Thailand and Bangladesh have been investigated for this purpose. A number of active compounds that affect TRAIL resistance were isolated from Amoora cucullata5 and Kandelia candel.6 The purification of active compounds from Combretum quadrangulare7 and Euphorbia neriifolia8 that enhanced DR5 promoter activity was reported. Some of these compounds overcome TRAIL resistance by up-regulating the expression of DR5.5,7 Six prenylated flavonoids, including three new compounds named pannokins A−C, were identified from Artocarpus champeden that potently exhibited the ability to overcome TRAIL resistance. Among these compounds, heterophyllin was suggested to induce © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The MeOH extract of A. communis roots was partitioned sequentially with hexane, EtOAc, n-BuOH, and water. Activityguided fractionation of the EtOAc-soluble fraction using silica gel, octadecylsilyl (ODS), Sephadex LH-20, and preparative ODS HPLC yielded compounds 1−12. The known compounds were identified as artonin E (5),11 morusin (6),12 artobiloxReceived: September 22, 2014

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Table 1. 1H and 13C NMR Spectroscopic Data for 1 and 2 1 position 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 1′ 2′ 3′ 4′ 5′ 6′ OH-5 OH-7 OH-2′ OH-4′ OH-5′

anthone (7),13 heterophyllin (8),14 cycloartobiloxanthone (9),13 cudraflavone B (10),15 artorigidin A14 (11), and pannokin B16 (12), respectively, by comparison of their spectroscopic data with values in the literature. Pannokin D (1) was isolated as an amorphous solid and shown to have the molecular formula C30H32O7 on the basis of its HRESIMS data (m/z 503.2096, calcd for C30H31O7, [M − H]−, Δ +2.6 mmu). Compound 1 gave the same molecular formula as heterophyllin (8),14 and the 1H and 13C NMR spectra of 1 (Table 1) were similar, which indicated this compound to be a flavonoid with two prenyl groups and a 2,2-dimethyl-2H-pyran ring. However, the 13C NMR chemical shift values for C-5, C-6, C-8, and C-8a (1: δC‑5 159.7, δC‑6 112.5, δC‑8 101.5, and δC‑8a 151.7) differed from those of 8 (8: δC‑5 155.9, δC‑6 106.2, δC‑8 112.4, and δC‑8a 156.4), suggesting that the substructure around ring A of these two compounds is different. Variations in the structures of 1 and 8 were identified as due to the presence of a prenyl side chain (C-14 to C-18) at C-6 and a 2,2-dimethyl-2Hpyran ring fused at C-7 and C-8 in 1 as opposed to the prenyl side chain at C-8 and the 2,2-dimethyl-2H-pyran ring fused at C-6 and C-7 in 8. These differences were evident from HMBC correlations from H-19 to C-7 and C-8a, from H-20 to C-8, and from H2-14 to C-5, C-6, and C-7 (Figure 1). Thus, pannokin D (1) was elucidated as shown. The HRESIMS data of pannokin E (2) exhibited a quasimolecular ion peak at m/z 597.2837 [M + Na]+ (calcd for C35H42O7Na, Δ +0.8 mmu). The 1H NMR spectrum of 2 (Table 1) was analogous to that of pannokin B (12), although its molecular formula included five more carbons and eight more hydrogens. The only major difference observed in the structure of 2 from that of pannokin B (12) was the presence of a geranyl group instead of the prenyl group in 12 at C-8. This was evident

a

δH (J in Hz)

3.14 d (7.1) 5.13 br t (7.1) 1.45 br s 1.56 br s 3.31 d (7.4) 5.22 br t (7.4) 1.79 br s 1.64 br s 6.61 d (10.0) 5.65 d (10.0) 1.45 br s 1.45 br s

6.58 s

6.84 s 13.67 s

2 δC

b

162.1 123.3 183.5 105.2 159.7 112.5 157.7 101.5 151.7 24.8 122.7 132.3 17.8 25.8 21.9 123.0 131.6 18.1 26.0 115.9 127.9 78.7 28.4 28.4

111.8 149.7 104.9 149.7 139.3 117.2

δH (J in Hz)

b

c

δH (J in Hz)

3.11 d (6.6) 5.14 m

3.00 d (7.0) 5.01 br t (7.0)

1.46 br s 1.67 br s 3.38 d (7.1) 5.22 br t (7.1)

1.52 br s 1.37 br s 3.29 5.12 br t (7.5)

1.80 br s 1.72 br s 3.41 d (7.1) 5.14 m

1.71 br s 1.60 br s 3.32 5.06 br t (6.8)

1.63 br s 2.02 m 2.00 m 5.02 br t (6.6)

1.50 br s 1.85 m 1.91 m 4.95 br t (6.7)

1.61 br s 1.55 br s

1.53 br s 1.45 br s

6.30 s

6.43 s

6.80 s

6.58 s 13.28 s 9.57 s 9.20 s 9.43 br s 8.45 br s

c

δC

161.5 119.1 181.9 103.7 156.0 111.0 158.6 105.9 153.0 23.0 121.8 131.1 17.4 25.5 21.4 122.6 130.7 17.8 25.6 21.4 122.0 134.4 15.7 39.0 26.1 124.1 130.7 17.5 25.5 111.1 148.4 103.7 148.4 137.9 116.3

a

Recorded in acetone-d6. bRecorded in CDCl3. cRecorded in DMSOd 6.

from 1H NMR signals, which were attributed to a geranyl group at δH 1.55 (3H, br s; H3-28), 1.61 (3H, br s; H3-27), 1.63 (3H, br s; H3-22), 2.00 (2H, m; H2-24), 2.02 (2H, m; H2-23), 3.41 (2H, d J = 7.1 Hz; H2-19), 5.02 (1H, br t J = 6.6 Hz; H-25), and 5.14 (1H, m; H-20). HMBC correlations (Figure 1) from H2-19 to C7, C-8, C-8a, and C-20, from H3-22 to C-21 and C-23, from H223 to C-20 and C-21, from H2-24 to C-23 and C-26, and from H3-27 and H3-28 to C-25 and C-26 supported the structure proposed. Thus, pannokin E (2) was elucidated as shown. Pannokin F (3), obtained as an amorphous solid, was shown to have the molecular formula C24H28O3 (m/z 363.1976, calcd for C24H27O3, [M − H]−, Δ +1.6 mmu) by HRESIMS. The 1H and 13 C NMR and HMQC data indicated the presence of a symmetrical 1,3,5-trisubstituted benzene ring [δH 6.52 (2H, d, J = 2.1 Hz)/δC 105.6 (2C) and δH 6.25 (1H, t, J = 2.1 Hz)/δC 102.6], a symmetrical tetrasubstituted benzene ring [δH 7.17 B

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Figure 1. Key HMBC correlations for compounds 1−4.

Figure 2. Effects of compounds 1−12, luteolin (positive control: Lut), and DMSO (negative control: Cont.) in the presence or absence of TRAIL on the viability of AGS cells. Cells were seeded in a 96-well culture plate (6 × 103 cells per well) for 24 h and then treated with the indicated concentrations of the compounds and TRAIL (100 ng/mL) for 24 h. Viability was evaluated by the FMCA method. Data represent the means ± SD (n = 3). The caspase inhibitor Z-VAD-FMK blocks cell death in AGS cells caused by the combined treatment with 5 (3 μM) and TRAIL (100 ng/mL). AGS cells were seeded in a 96-well culture plate (6 × 103 cells per well) for 24 h and then treated with the indicated reagent and concentrations. Data represent the means ± SD (n = 3).

(2H, s)/δC 126.5 (2C)], a disubstituted E-double bond [δH 6.97 (1H, d, J = 16.3 Hz)/δC 129.1 and δH 6.83 (1H, d, J = 16.3 Hz)/ δC 126.7], and two prenyl side chain moieties [δH 1.73 (12H, br s)/δC 25.9 (2C) and 17.9 (2C), δH 5.34 (2H, br t, J = 7.3 Hz)/δC 123.4 (2C), δH 3.36 (4H, br d, J = 7.3 Hz)/δC 28.8 (2C), and δC 133.0 (2C)]. HMBC correlations (Figure 1) from H2-7 to C-2 and C-4, which were equivalent to those from H2-12 to C-6 and C-4, indicated that two prenyl moieties are attached to C-3 and C-5, leading to the construction of a symmetrical prenylated benzene ring (substructure A). The symmetrical properties of substructure A were supported by the HMBC correlation from H-2 (H-6) to C-6 (C-2). The HMBC correlation from H-2′ and H-6′ to C-β suggested the connection of the 1,3,5-trisubstituted benzene ring and trans-double bond for the construction of substructure B. The connectivity was established between substructures A and B by HMBC correlations from H-α and H-β to C-1 and from H-2 and H-6 to C-α. The HMBC correlations obtained were used to assign all the 1H and 13C

NMR signals for compound 3. An assessment of the molecular formula and deshielded chemical shift values of C-4 (δC 153.2) and C-3′ and C-5′ (δC 159.6) indicated the presence of hydroxy groups at these positions. Thus, the structure of pannokin F was elucidated as 3, a resveratrol derivative. Pannokin G (4), isolated as an amorphous solid, was shown to have the molecular formula C25H30O3 by HRESIMS (m/z 393.2055, calcd for C25H29O3, [M − H]−, Δ −1.1 mmu), with one more carbon and two more hydrogens than 3. The 1H and 13 C NMR data closely resembled those of the latter compound, except for an additional methoxy group [δH 3.70 (3H, s)/δC 62.1] instead of one less aromatic methine signal observed in 3. The position of the methoxy group was assigned at C-2 by the HMBC correlation from H3CO, H-α, and H2-7 to C-2. Thus, the structure of pannokin G (4) was concluded to be the 2-methoxy derivative of 3. The ability of the isolated compounds (1−12) to overcome TRAIL resistance was evaluated using AGS, a human gastric C

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mediate apoptotic cell death; therefore, it was investigated as to whether 5 induced apoptosis using flow cytometry. In this evaluation, cells were stained with annexin V-FLUOS, a phospholipid-binding protein that has a high affinity to cell surface phosphatidylserine, and propidium iodide (PI), to detect apoptosis. Although the treatment with either TRAIL or 5 alone did not induce significant changes, the combined treatment with 5 (3 μM) and TRAIL (100 ng/mL) for 6 h increased the population of apoptotic cells (32.7%, lower right) significantly. Furthermore, the population of apoptotic cells induced by the combined treatment with 5 and TRAIL was reduced (from 32.7% to 2.9%) in the presence of Z-VAD-FMK, a pan-caspase inhibitor, indicating that the combined treatment with 5 and TRAIL induced apoptosis in a caspase-dependent manner (Figure 4). Artonin E (5) at a concentration of 3 and 5 μM enhanced caspase 3/7 activity by 3.7- and 11.1-fold, respectively (Figure 5). Moreover, this activity was 12.2- and 34.3-fold higher

adenocarcinoma cell line that exhibits TRAIL resistance. As shown in Figure 2, the treatment of AGS cells with 100 ng/mL TRAIL for 24 h resulted in only a slight decrease in cell viability (9%), while luteolin17 at 17.5 μM, which was used as a positive control, was 44% more potent when administered in combination with TRAIL than TRAIL alone. The cell viabilities of each compound (1−12) were examined at several concentrations, and it was revealed that the treatment of AGS cells with 1 (2 μM), 2 (5 μM), 3 (20 μM), 4 (40 μM), 5 (3 μM), 6 (40 μM), 7 (10 μM), 8 (2 μM), 10 (40 μM), 11 (7.5 μM), and 12 (7.5 μM) in the presence of TRAIL led to cell viabilities 46%, 30%, 25%, 40%, 44%, 25%, 32%, 31%, 27%, 26%, and 30% lower, respectively, than that of AGS cells treated with each compound alone (without TRAIL). These results confirmed the ability of these compounds to overcome TRAIL resistance. Compounds 1 (2 μM), 5 (3 μM), and 8 (2 μM) in overcoming TRAIL resistance were more potent than the other compounds tested since they exhibited larger differences (31−46%) in cell viability at lower concentrations (2−3 μM). Artonin E (5), which exhibited potent activity, was selected for further studies using AGS cells, since a sufficient quantity of the compound was isolated for the purpose. To investigate the involvement of apoptosis in the TRAIL-resistance-overcoming activity of 5, the effects of caspases were evaluated using the pancaspase inhibitor, Z-VAD-FMK. As shown in Figure 3, the

Figure 5. Effects of the combined treatment with 5 and TRAIL on caspase 3/7 activity in AGS cells. Data are presented as the means ± SD (n = 3). Significance was determined with Tukey’s test, **p < 0.01.

following the treatment with 5 at concentrations of 3 and 5 μM in combination with TRAIL, respectively, than the treatment with 5 alone. These results indicated that the combined treatment with 5 and TRAIL induced apoptotic cell death in a caspasedependent manner. In the TRAIL signaling pathway, the binding of TRAIL to death receptors (DR) initiates the signal cascade.1 p53, an important tumor suppressor protein that participates in cellular responses to various stresses, acts as a transcriptional factor of the proteins involved in the TRAIL signal.18 A previous study has suggested that enhancements in the expression of DR5 are mediated by p53.19 Accordingly, the protein levels of p53 and DR5 were investigated by Western blot analysis in the present

Figure 3. Caspase inhibitor Z-VAD-FMK blocks cell death in AGS cells caused by the combined treatment with 5 (3 μM) and TRAIL (100 ng/ mL). AGS cells were seeded in a 96-well culture plate (6 × 103 cells per well) for 24 h and then treated with the indicated reagent and concentrations. Data represent the means ± SD (n = 3). Significance was determined with Tukey’s test, **p < 0.01.

decrease observed in cell viability by the combined treatment with 5 and TRAIL was abolished in the presence of Z-VADFMK. This result indicated that the TRAIL-resistance-overcoming activity of 5 is caspase-dependent. Caspases typically

Figure 4. Induction of apoptosis by the combined treatment with 5 (3 μM) and TRAIL (100 ng/mL) and its inhibition by the caspase inhibitor Z-VADFMK (50 μM). Attached cells and floating cells were collected and stained using the annexin-V-FLOUS staining kit (Roche). Stained cells were immediately analyzed by flow cytometry (Millipore, Guava easyCyte). The numbers on the right lower side indicate the percentage of apoptotic cells. D

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study. As shown in Figure 6, the treatment of AGS cells with 5 (3 μM) increased p53 and DR5 levels after 6 h.

Figure 6. p53 and DR5 protein levels in AGS cells treated with 5 (3 μM) for 1.5, 3, and 6 h as analyzed by Western blotting.

Previous studies have reported that ROS (reactive oxygen species) enhance the expression of DR5 and p53 in order to sensitize the TRAIL resistance in TRAIL-resistant cancer cells upstream of this signaling pathway.20,21 N-Acetylcysteine (NAC), which scavenges ROS, was employed to investigate the involvement of ROS in the effects of 5. As shown in Figure 7,

Figure 9. Compound 5 (3 μM) depolarizes mitochondrial membrane potential in AGS cells. AGS cells (1 × 105 cells/well) were seeded in 12well plates. After a 24 h preincubation, cells were treated with artonin E (5, 3 μM) for 6 h. Attached cells and floating cells were collected and stained using the Guava EasyCyte Mitopotential kit (Merck Millipore). Stained cells were immediately analyzed by flow cytometry (Millipore, Guava easyCyte). Numbers on the right indicate the percentage of depolarized cells.

Figure 7. Pretreatment with NAC (1 mM) inhibits cell death caused by both 5 alone and the combined treatment with 5 and TRAIL (100 ng/ mL) (cont. = control, Lut = luteolin). AGS cells were seeded in a 96-well culture plate (6 × 103 cells per well) for 24 h and then treated with the indicated reagent and concentrations. Data represent the means ± SD (n = 3). Significance was determined with Tukey’s test, *p < 0.05, **p < 0.01.

pretreatment of NAC abolished the decreases observed in cell viability by both 5 and the combination of 5 and TRAIL. This indicated the involvement of ROS in the cytotoxicity of both 5 alone and the combination of 5 and TRAIL. As shown in Figure 8, although 5 increased the protein levels of DR5 by 1.9-fold,

Figure 10. TRAIL-resistance-overcoming activity by artonin E (5).

the TRAIL signaling pathway, together with the extrinsic pathway in which caspase-8 directly activates caspase-3.1 Decreases in mitochondrial membrane potential have also been implicated in apoptotic cell death. The mitochondrial membrane potential was next evaluated using JC-1 staining. JC-1 is a fluorescent and cationic dye that aggregates and accumulates in mitochondria in a mitochondrial membrane potentialdependent manner. On the other hand, the JC-1 dye leaks into the cytosol of apoptotic cells when the membrane potential is depolarized and where it exists as a monomer with green fluorescence. As shown in Figure 9, treatment with artonin E (5) increased the population of cells displaying strong green fluorescence, indicating that 5 decreases mitochondrial membrane potential. These results suggest that 5 also activated the intrinsic apoptosis pathway (Figure 10). In a previous study, heterophyllin (8), which was also isolated in the present investigation, was suggested to have induced a CHOP-dependent up-regulation of DR5 expression, resulting in apoptosis in AGS cells.9 A previous study reported that ROS modulated TRAIL signals by enhancing DR5 expression

Figure 8. Pretreatment with NAC (1 mM) inhibits increases in DR5 protein levels by 5 (3 μM).

pretreatment with NAC prevented these increases in DR5 by 5. These results suggested that 5 enhances DR5 protein levels in a ROS-dependent manner. Taken together, artonin E (5) may abrogate TRAIL resistance by inducing ROS- and p53-mediated increased levels in DR5 (Figure 10). The presence of an intrinsic (mitochondrial) apoptosis pathway has also been reported downstream of caspase-8 in E

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upstream of CHOP.20 Collectively, artonin E (5) and heterophyllin (8) may be presumed to be responsible for most of the effects on the TRAIL signaling pathway by the A. communis root extract investigated. Artonin E (5) has been shown to sensitize lung cancer cells to anoikis, which is mediated by the down-regulation of MCL1,22 and to have antimicrobial activity.23 However, to the best of our knowledge, this is the first study on the ability of artonin E (5) to overcome TRAIL resistance.

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fractions 5A to 5D. Fraction 5B (99.6 mg) was further purified by preparative HPLC [YMC-Pack ODS-AM, 10 × 250 mm; 74:26 MeOH−H2O; flow rate: 2.0 mL/min; RI and UV detection at 254 nm] to give 5 (55.7 mg, tR 26 min). Fraction 5C (36.5 mg) was further purified by preparative HPLC [YMC-Pack ODS-AM, 10 × 250 mm; 71:29 MeOH−H2O; flow rate: 2.0 mL/min; RI and UV detection at 254 nm] to give 7 (8.3 mg, tR 42 min). Fraction 2G (74 mg), eluted with 5:1 MeOH−H2O, was subjected to Sephadex LH-20 column chromatography (16 × 600 mm), eluted with MeOH, to afford fractions 6A to 6G. Fraction 6B (20.3 mg) was further purified by preparative HPLC [YMCPack ODS-AM, 10 × 250 mm; MeOH−H2O (77:23); flow rate: 2.0 mL/min; RI and UV detection at 254 nm] to give 7 (5.7 mg, tR 26 min). Fraction 1A (377 mg), eluted with 13:1 CHCl3−MeOH, was subjected to ODS column chromatography (22 × 210 mm) using MeOH−H2O solvent mixtures of progressively decreasing polarity to afford fractions 7A to 7M. Fraction 7E (28.2 mg), eluted with 7:1 MeOH−H2O, was subjected to preparative HPLC [YMC-Pack ODS-AM, 10 × 250 mm; 79:21 MeOH−H2O; flow rate: 2.0 mL/min; RI and UV detection at 254 nm] to give 6 (1.3 mg, tR 30 min), 1 (3.6 mg, tR 35 min), and a fraction (13.4 mg, tR 25 min) that was further separated by preparative HPLC [Develosil ODS HG-5, 10 × 250 mm; MeOH−H2O (72:28); flow rate: 2.0 mL/min; RI and UV detection at 254 nm] to give 5 (4.9 mg, tR 32 min) and 2 (4.1 mg, tR 40 min). Pannokin D (1): amorphous solid; UV (MeOH) λmax (log ε) 367 (3.8), 341 (3.8), 269 (4.3), 234 (4.3) nm; IR (ATR) νmax 3395, 2973, 1615 cm−1; 1H and 13C NMR (Table 1); HRESIMS m/z 597.2837 [M + Na]+ (calcd for C35H42O7, 597.2828). Pannokin E (2): amorphous solid; UV (MeOH) λmax (log ε) 367 (3.8), 268 (4.1) nm; IR (ATR) νmax 3348, 2986, 1644 cm−1; 1H and 13C NMR (Table 1); HRESIMS m/z 597.2837 [M + Na]+ (calcd for C35H42O7, 597.2828). Pannokin F (3): amorphous solid; UV (MeOH) λmax (log ε) 310 (4.2) nm; IR (ATR) νmax 3370, 2116, 1635 cm−1; 1H and 13C NMR (Table 2); HRESIMS m/z 363.1976 [M − H]− (calcd for C24H27O3, 363.1960)

EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were measured using a Shimadzu UV mini-1240 spectrometer. IR spectra were measured using the ATR (attenuated total reflection) method with a JASCO FT-IR 230 spectrophotometer. NMR spectra were recorded on JEOL ECP600, ECP400, and ECS400 NMR spectrometers with a deuterated solvent, the chemical shifts of which were used as an internal standard. HRESIMS were obtained on a JEOL JMS-T100LP mass spectrometer. Column chromatography was performed using silica gel PSQ100B, Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai, Japan), and Sephadex LH-20 (GE Healthcare, Little Chalfont, UK). Preparative HPLC was performed using YMC-Pack ODS-AM (YMC Co., Ltd., Kyoto, Japan), Develosil ODS-HG-5 (Nomura Kagaku, Seto, Japan), and Phenomenex Luna 5 μm phenyl-hexyl (Phenomenex, Inc., Torrance, CA, USA). Plant Material. The roots of Artocarpus communis were collected at Khon Kaen, Thailand, in December 2009 and were identified taxonomically by T.K. A voucher specimen (KKP286) was deposited at the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University. Extraction and Isolation. The air-dried and ground roots of A. communis (117 g) were subjected to extraction with MeOH for 2 days at room temperature, and then homogenization and filtration, followed by evaporation and vacuum desiccation, were used to obtain a crude extract (9.6 g). This extract was suspended in 10% aqueous MeOH (130 mL) and partitioned between hexane, EtOAc, and BuOH (130 mL × 3) to obtain the corresponding dried extracts. The EtOAc extract (4.5 g) was subjected to silica gel PSQ100B column chromatography (24 × 470 mm) using CHCl3−MeOH solvent systems of increasing polarity to afford fractions 1A to 1G. Fraction 1B (784 mg), eluted with 11:1 CHCl3−MeOH, was subjected to ODS column chromatography (22 × 240 mm) using MeOH−H2O solvent mixtures of progressively decreasing polarity to afford fractions 2A to 2L. Fraction 2H (50 mg), eluted with 5:1 MeOH−H2O, was subjected to Sephadex LH-20 column chromatography (16 × 620 mm), eluted with MeOH, to afford fractions 3A to 3D, and fraction 3D (3.1 mg) was identified as 9. Fraction 3B (21 mg) was subjected to preparative HPLC [Develosil ODS-HG-5, 10 × 250 mm; 80:20 MeOH−H2O; flow rate: 2.0 mL/min; RI and UV detection at 254 nm] to give 8 (2.3 mg, tR 30 min), 2 (3.8 mg, tR 35 min), and 1 (3.1 mg, tR 39 min). Fraction 3C (14.3 mg) was subjected to preparative HPLC [Develosil ODS-HG-5, 10 × 250 mm; 82:18 MeOH−H2O; flow rate: 2.0 mL/min; RI and UV detection at 254 nm] to give 6 (1.0 mg, tR 32 min). Fraction 2F (151 mg), eluted with 4:1 MeOH−H2O, was subjected to Sephadex LH-20 column chromatography (16 × 620 mm) eluted with MeOH to afford fractions 4A to 4D. Fraction 4B (62.5 mg) was subjected to preparative HPLC [YMC-Pack ODS-AM, 10 × 250 mm; 72:28 MeOH−H2O; flow rate: 2.0 mL/min; RI and UV detection at 254 nm] to give 5 (35.7 mg, tR 30 min), 4 (2.3 mg, tR 41 min), and a fraction (6.6 mg, tR 56 min), which was further purified by preparative HPLC [Phenomenex Luna 5 μm phenyl-hexyl, 10 × 250 mm; MeOH−H2O (78:22); flow rate: 2.0 mL/min; RI and UV detection at 254 nm] to afford 12 (0.5 mg, tR 32 min) and 11 (1.4 mg, tR 40 min). Fraction 4C (26.7 mg) was subjected to preparative HPLC [Phenomenex Luna 5 μm phenyl-hexyl, 10 × 250 mm; MeOH−H2O (72:28); flow rate: 2.0 mL/min; RI and UV detection at 254 nm] to give 3 (1.3 mg, tR 42 min) and 7 (5.8 mg, tR 50 min). Fraction 2E (199 mg), eluted with 4:1 MeOH−H2O, was subjected to Sephadex LH-20 column chromatography (16 × 650 mm) eluted with MeOH to afford

Table 2. 1H and 13C NMR Spectroscopic Data in Acetone-d6 for 3 and 4 3 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 α β 1′ 2′, 6′ 3′, 5′ 4′ OMe-2 OH-4 OH-3′,5′ F

δH (J in Hz) 7.17 s

7.17 s 3.36 br d (7.3) 5.34 br t (7.3) 1.73 br s 1.73 br s 3.36 br d (7.3) 5.34 br t (7.3) 1.73 br s 1.73 br s 6.97 d (16.3) 6.83 d (16.3) 6.52 d (2.1) 6.25 t (2.1) 7.17 s 8.16 br s

4 δC 140.9 126.5 129.5 153.2 129.5 126.5 28.8 123.4 133.0 17.9 25.9 28.8 123.4 133.0 17.9 25.9 129.1 126.7 130.2 105.6 159.6 102.6

δH (J in Hz)

7.30 s 3.41 d (6.9) 5.21 br t (6.9) 1.78 br s 1.65 br s 3.37 d (7.2) 5.34 br t (7.2) 1.73 br s 1.73 br s 7.22 d (16.4) 6.88 d (16.4) 6.54 d (2.2) 6.26 t (2.2) 3.70 s 7.14 br s 8.21 br s

δC 125.3 156.3 125.3 154.2 122.6 123.4 23.9 123.1 132.0 17.9 25.8 29.1 124.2 133.1 18.0 25.9 124.2 127.8 141.2 105.6 159.7 102.7 62.1

DOI: 10.1021/np500734t J. Nat. Prod. XXXX, XXX, XXX−XXX

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Pannokin G (4): amorphous solid; UV (MeOH) λmax (log ε) 312 (3.9) nm; IR (ATR) νmax 3359, 2977, 1595 cm−1; 1H and 13C NMR (Table 2); HRESIMS m/z 393.2055 [M − H]− (calcd for C24H29O4, 393.2066). Cell Cultures. AGS cells were obtained from the Institute of Development, Aging and Cancer, Tohoku University. Cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Wako, Osaka, Japan) with 10% FBS. All cultures were maintained in a humidified incubator at 37 °C in 5% CO2/95% air. Viability Assay (FMCA). Cell viability was assessed in the presence and absence of TRAIL using TRAIL-resistant human gastric adenocarcinoma (AGS) cells.24 AGS cells were seeded in a 96-well culture plate (6 × 103 cells per well) in 200 μL of RPMI medium containing 10% FBS. They were then incubated at 37 °C in a 5% CO2 incubator for 24 h. Test samples at different doses with or without TRAIL (100 ng/mL) were added to each well. After a 24 h incubation, the cells were washed with PBS, and 200 μL of PBS containing fluorescein diacetate (10 μg/mL)25 was added to each well. The plates were incubated at 37 °C for 1 h, and fluorescence at 538 nm with excitation at 485 nm was measured using Fluoroskan Ascent (Thermo Fisher Scientific, Waltham, MA, USA). Luteolin (purity ≥98%, SigmaAldrich) was used as a positive control. Apoptosis Assay. AGS cells (1 × 105 cells/well) were seeded in 12well plates and, after 24 h, were treated with artonin E (5, 3 μM) for 6 h in either the absence or presence of TRAIL (100 nM) or Z-VAD-FMK (50 μM). Attached cells were harvested by trypsinization and centrifuged (1000 rpm, 5 min) with the floating cells in medium, then stained using the annexin-V-FLOUS staining kit (Roche, Basel, Switzerland), according to the manufacturer’s protocol. Stained cells were then immediately analyzed by flow cytometry (Guava easyCyte, Merck Millipore, Billerica, MA, USA), and data were analyzed with the GuavaSoft software. Apoptotic cells with exposed phosphatidylserine, but intact cell membranes bound to annexin-V-fluorescein and excluded propidium iodide. Cells in necrotic stages were labeled with both annexin-V-fluorescein and propidium iodide. A minimum of 5000 events were collected for each treatment. All experiments were performed in triplicate. Mitochondrial Potential Assay. AGS cells (1 × 105 cells/well) were seeded in 12-well plates and, after 24 h, were treated with artonin E (5, 3 μM) for 6 h. Attached cells were harvested by trypsinization and centrifuged (1000 rpm, 5 min) with the floating cells in medium, then stained using the Guava EasyCyte Mitopotential kit (Merck Millipore) according to the manufacturer’s instructions. Stained cells were then immediately analyzed by flow cytometry (Millipore, Guava easyCyte), and data were analyzed with the GuavaSoft software. A minimum of 5000 events were collected for each treatment. All experiments were performed in triplicate. Caspase-3/7 Activity. Caspase-3/7 activity was measured using the Caspase-Glo 3/7 Assay kit (Promega, Fitchburg, WI, USA) according to the manufacturer’s protocol, with several modifications. AGS cells (6 × 104 cells/well) were seeded in 96-well plates and cultivated for 24 h. After being incubated with 3 and 5 μM of 5 with or without TRAIL (100 ng/mL) for 6 h, cells were washed with PBS; then 120 μL/well of PBS containing fluorescein diacetate (10 μg/mL)25 was added. After a 1 h incubation, viability, as a primary assay, was evaluated using the abovementioned protocol. Caspase-Glo 3/7 buffer (45 μL/well) was added to the PBS with fluorescein diacetate and shaken for 30 min at room temperature. Then, 4×Caspase-Glo 3/7 reagent (15 μL/well) was added and shaken for 30 min at room temperature in the dark, and chemiluminescence was monitored using Luminoskan Ascent (Thermo Fisher Scientific). Caspase activity was normalized with viability. Western Blot Analysis. The lysates of AGS cells were prepared using lysis buffer [Tris·HCl (20 mM, pH 7.4), NaCl (150 mM), Triton X-100 (0.5%), sodium deoxycholate (0.5%), EDTA (10 mM), sodium orthovanadate (1 mM), and NaF (0.1 mM)], containing protease inhibitor cocktail (1%; #25955-11, Nacalai Tesque, Tokyo, Japan). Proteins were separated on a 12.5% SDS-polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane (BioRad Laboratories, Hercules, CA, USA). After blocking with 5% skimmed milk in Tris-buffered saline with 1% Tween-20 (TBST) for 1 h at room

temperature, the membrane was incubated at room temperature with primary antibodies [anti-p53 (1:4000, #P5813, Sigma-Aldrich, St. Louis, MO, USA); anti-DR5 (1:750, #D3938, Sigma-Aldrich); and anti-β-actin (1:4000, #A2228, Sigma-Aldrich) overnight at room temperature]. The anti-β-actin antibody served as an internal control. After being washed with TBST, the membrane was incubated with either horseradish peroxidase conjugated anti-mouse IgG (1:4000, #NA931VS, GE Healthcare) or anti-rabbit (1:4000, #705-035-003, Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature. After further washing with TBST, immunoreactive bands were detected using the ECL Advanced Western detection system (GE Healthcare) or Immobilon Western chemiluminescent HRP substrate (Merck Millipore) using Molecular Imager ChemiDoc XRS+ (Bio-Rad Laboratories).

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ASSOCIATED CONTENT

* Supporting Information S

This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-43-226-2923. Fax: +81-43-226-2923. E-mail: mish@ chiba-u.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by KAKENHI Grant Number 23102008 from MEXT, 26305001 and 25870128 from JSPS, the Cosmetology Research Foundation, the Hamaguchi Foundation for the Advancement of Biochemistry, and the Uehara Memorial Foundation.

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