Boesenberols, Pimarane Diterpenes with TRAIL ... - ACS Publications

Aug 10, 2016 - ABSTRACT: TRAIL is a potent and selective inducer of apoptosis in most cancer cells while sparing normal cells, which makes it an attra...
2 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/jnp

Boesenberols, Pimarane Diterpenes with TRAIL-ResistanceOvercoming Activity from Boesenbergia pandurata Utpal K. Karmakar,†,‡ Naoki Ishikawa,† Midori A. Arai,† Firoj Ahmed,†,§ Takashi Koyano,⊥ Thaworn Kowithayakorn,∥ and Masami Ishibashi*,† †

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Pharmacy Discipline, Life Science School, Khulna University, Khulna-9208, Bangladesh § Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka-1000, Bangladesh ⊥ 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: TRAIL is a potent and selective inducer of apoptosis in most cancer cells while sparing normal cells, which makes it an attractive target for the development of new cancer therapies. In a screening program on natural resources with the ability to abrogate TRAIL resistance, the bioassayguided fractionation of Boesenbergia pandurata rhizomes resulted in the isolation of 17 pimarane diterpenes and a monoterpene. Among these, compounds 1−8, named boesenberols A−H, are new pimarane diterpenes. All compounds exhibited TRAIL-resistance-overcoming activity in TRAIL-resistant AGS cells. Subtoxic doses of the major compound 9 sensitized AGS cells to TRAIL-induced apoptosis by up-regulating apoptosis-inducing proteins, such as DR4, DR5, p53, Fas, CHOP, Bak, and cleaved caspases-3, -8, and -9, and down-regulating the levels of cell survival proteins, such as Bcl-2, cFLIP, and GSK-3β, in TRAIL-resistant AGS cells. Furthermore, compound 9 did not decrease the viability of noncancerous (HEK293) cells at concentrations up to 30 μM.

T

translocates to mitochondria and activates mitochondrial pathways.4 In mitochondrial pathways, cleaved Bid, called truncated Bid (tBid), translocates to mitochondria, in which it interacts with other proapoptotic members of the Bcl-2 family, Bax and Bak, forming pores in the outer mitochondrial membrane, causing the release of proapoptogenic factors such as cytochrome c. Cytochrome c then binds to the adaptor apoptotic protease activating factor 1 (Apaf-1) and pro-caspase9 in order to form an apoptosome, which results in the activation of caspase-9. Activated caspase-9 serves as initiator caspase to activate executioner caspases-3, -6, and -7, which cause cell death.5 However, large numbers of cancer cells, particularly some highly malignant tumors, have developed resistance to TRAILinduced apoptosis.4 For example, lung, breast, prostate, and gastric cancers are considered to be resistant to TRAIL. TRAIL resistance may develop at different points in the signaling pathways of TRAIL-induced apoptosis. Decreases in the expression of the death receptors DR4 and DR5 due to mutations may lead to TRAIL resistance. The adaptor protein FADD and caspase-8 are essential for forming the deathinducing signaling complex, and defects in either of these

umor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily with 20 kDa and 281 amino acids containing a type II transmembrane protein.1 TRAIL is perceived as a promising chemotherapeutic approach because it specifically targets cancer cells. It is essential for immune surveillance, the apoptosis of unwanted cells, and the suppression of spontaneous tumor formation.2 Apoptosis is a genetically controlled mechanism that is important for the maintenance of tissue homeostasis, proper development, and the elimination of genetically transformed cells. Apoptosis induces distinct morphological and biochemical changes in cells that are mediated by caspases.3 There are two essential apoptotic pathways, referred to as the death-receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway. TRAIL has the ability to activate either pathway depending on the cell type. TRAIL-induced apoptosis is initiated after the binding of TRAIL to death receptors (DR4 and DR5), which then homotrimerize and transmit a death signal to the cytoplasm, thereby enabling the recruitment of the adaptor protein Fas-associated death domain (FADD), formation of the death-inducing signaling complex (DISC), proteolytic activation of caspase-8, and consequently the activation of caspase-3, resulting in apoptosis. Proteolyticactivated caspase-8 further activates Bid, which, in turn, © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 11, 2016

A

DOI: 10.1021/acs.jnatprod.6b00424 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

molecules may lead to TRAIL resistance.5 The overexpression of Bcl-2 or Bcl-X(L), the loss of Bax or Bak function, and the strong expression of inhibitors of apoptosis proteins, c-FLIP, and GSK-3β have also been reported to result in TRAIL resistance.6,7 The identification of cellular targets and a deeper understanding of the mechanisms responsible for natural compounds that selectively act on malignant cells are, thus, very important for overcoming TRAIL resistance and anticancer drug discovery. In a screening program, the TRAIL-resistance-overcoming activities of extracts from organisms were assessed by comparing the cell viabilities of TRAIL-resistant human gastric adenocarcinoma (AGS) cell lines in the presence and absence of TRAIL.3 A 25% difference in viability between the presence and absence of TRAIL indicates that the compound overcomes TRAIL resistance, while a greater difference in viability indicates strong TRAIL-resistance-overcoming activity.8 After the initial screening of the extract library, a MeOH extract of Boesenbergia pandurata Schltr. (Zingiberaceae) rhizomes was found to actively overcome TRAIL resistance. B. pandurata is a culinary herb found in Southeast Asia and mainland China. According to Indonesian medicinal plant literature, the fresh rhizomes of B. pandurata have long been utilized as a spice to season vegetables.9 These rhizomes have been used traditionally in the treatment of dry cough, stomach ulcers, inflammation of the uterus, vaginal infections, and cancer.9 The rhizomes of the plant represent one of the components of the herbal medicine system “jamu” in Indonesia.10 Several flavonoid chalcones, flavanones, flavones, and pimarane diterpenes have been isolated and identified from the rhizome extract of B. pandurata.9 The bioassay-guided fractionation of the MeOH extract of rhizomes led to the isolation of 17 pimarane diterpenes and one monoterpene. Among these, compounds 1−8, named boesenberols A−H, are new pimarane diterpenes. This report describes not only the isolation, structural elucidation, and TRAIL-resistance-overcoming activity of isolated compounds in TRAIL-resistant AGS cells but also the sensitizing effect of compound 9 on TRAIL-induced apoptosis through the up-regulation of DR4, DR5, Fas, p53, CHOP, and Bax following the activation of caspases-8, -9, and -3 and also the down-regulation of the cell survival proteins Bcl2, c-FLIP, and GSK-3β in TRAIL-resistant AGS cells.

activity-guided fractionation of the EtOAc-soluble fraction using silica gel, octadecylsilyl (ODS), and preparative reversed-phase HPLC yielded compounds 1−18. Compounds 1−8 are new pimarane diterpenes. The known compounds were identified as 6β-acetoxysandaracopimaradiene-1α,9α-diol (9),11 sandaracopimaradiene-1α,6β,9α-triol (10),11 kaempulchraol C (11),12 sandaracopimaradiene-1α,2α-diol (12),13 sandaracopimaradiene-6β,9α-diol-1-one (13),11 6β-acetoxysandaracopimaradiene-9α-ol-1-one (14),11 sandaracopimaradiene1α,9α-diol (15),11 kaempulchraol H (16),12 1,11α-dihydroxypimara-8(14)-15-diene (17),14 and 5α-hydroxy-2-oxo-pmenth-6(1)-ene (18),15 respectively, by comparison of their spectroscopic data in the literature (Chart S1, Supporting Information). Boesenberol A (1) was isolated as a colorless, amorphous solid, and its molecular formula was determined by HRESIMS as C20H30O3 (MW 318). The IR spectrum of 1 showed an absorption band due to hydroxy and carbonyl groups at 3470 and 1651 cm−1 respectively. The 13C NMR (Table 1) spectrum revealed 20 signals including four methyls (δC 24.2, 23.9, 34.7, and 21.5), five sp3 methylenes (δC 26.8, 36.7, 36.1, 22.3, and 36.6), three sp3 methines including two oxygenated methines (δC 73.4, 46.6, and 65.1), one sp2 methylene (δC 114.8), one sp2 methine (δC 141.9), one carbonyl (δC 204.6), three sp3 quaternary carbons (δC 33.8, 45.0, and 48.6), and two sp2 quaternary carbons (δC 128.7 and 166.4). The 1H NMR (Table 2) spectrum displayed signals due to a terminal vinyl group [δH 5.90 (dd, J = 10.8, 17.4 Hz, H-15), 5.04 (d, J = 10.8 Hz, H-16a), 4.94 (d, J = 17.4 Hz, H-16b)], two oxygenated methines [δH 3.81 (t, J = 1.8 Hz, H-1) and 4.55 (br d, J = 4.8 Hz, H-6)], and four tertiary methyls [δH 1.15 (s, H3-17), 1.23 (s, H3-18), 1.01 (s, H3-19), and 1.43 (s, H3-20)]. The aforementioned spectroscopic data were consistent with a pimarane diterpenetype compound,16 which was confirmed by HMBC correlations. Specifically, HMBC correlations (Figure S1 and Table S1, Supporting Information) from H3-17 to C-12, C-13, C-14, and C-15, from H-16 to C-13, and from H-12 to C-13 and C-15 indicated that a methyl group and vinyl unit are connected to C-13 and a carbonyl group is present at C-14. HMBC correlations from H3-20 to C-1 and C-9, from H-6 to C-10 and C-8, and from H-1 to C-3 and C-5 suggested hydroxy groups as being present at C-1 and C-6, with a double bond located between C-8 and C-9. The relative configuration of 1 was determined by comparing its spectroscopic data with these of the known compound kaempulchraol G12 and by analysis of coupling constants and NOE correlations. The small coupling constant for H-1 [δH 3.81 (t, J1,2α = 1.8 Hz and J1,2β = 1.8 Hz)] indicated this proton to be in the equatorial position, and, thus, the hydroxy group substituent at C-1 is α-axially oriented. The H-1 and H-20 protons also showed NOE correlations, and, thus, the methyl at C-10 is β-axially oriented. The H-5 and H-6 protons displayed NOE correlations and had a small coupling constant between them since H-5 appeared as a broad singlet. The H-6 signal was observed at δH 4.55 (br d, J = 4.8 Hz). These observations are similar to those observed for the corresponding positions of other pimarane diterpenes such as kaempulchraol G,12 with a 6β-hydroxy group. Therefore, the structure of compound 1 was proposed as 1α,6β-dihydroxypimara-8,15-dien-14-one and named boesenberol A. Boesenberol B (2) was obtained as a colorless, amorphous solid, and its molecular formula was assigned by HRESIMS as C20H32O3 (MW 320). The IR spectrum revealed absorption bands at 3387 cm−1 ascribable to hydroxy groups. The 1H and

■

RESULTS AND DISCUSSION A MeOH extract of B. pandurata rhizomes was partitioned sequentially in hexane, EtOAc, n-BuOH, and water. The B

DOI: 10.1021/acs.jnatprod.6b00424 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 13C NMR Spectroscopic Data (150 MHz, CDCl3) of Bosenberols A−H (1−8) (δ in ppm) position

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

73.4 26.8 36.7 33.8 46.6 65.1 36.1 128.7 166.4 45.0 22.3 36.6 48.6 204.6 141.9 114.8 24.2 23.9 34.7 21.5

73.7 26.2 33.6 32.6 34.0 30.1 74.0 136.0 78.6 43.1 26.0 30.6 37.7 137.0 147.5 111.2 23.0 22.5 33.6 18.1

75.4 26.5 35.5 33.4 37.2 73.8 75.2 133.0 79.0 43.8 26.5 31.0 37.9 139.8 147.2 111.5 23.2 24.8 33.6 21.2 170.2 21.7

33.7 43.7 18.6 33.8 41.7 73.4 75.6 134.0 76.0 42.8 26.4 31.4 38.1 140.1 147.2 111.4 23.5 24.0 33.6 20.8 170.4 21.6

33.9 18.7 41.2 33.1 42.4 32.6 69.8 139.9 75.8 42.9 66.2 40.7 37.8 127.3 147.8 111.3 25.1 22.2 33.9 17.9

39.1 19.0 42.8 33.7 49.1 71.8 71.6 125.6 146.9 38.1 21.3 29.7 40.0 81.8 143.9 112.7 23.5 24.1 33.4 21.8 62.0

39.5 19.1 42.5 33.7 49.0 67.0 86.2 123.8 147.3 38.0 20.2 27.5 39.9 88.2 146.2 111.6 20.6 24.4 33.1 22.4 57.4 60.4

39.1 19.0 42.6 33.6 49.3 66.6 80.8 124.4 146.6 37.7 21.3 30.0 40.1 81.8 144.0 112.6 23.2 24.1 33.1 21.8 57.8 61.9

13

an acetoxy group is located at C-6 (Figure S1 and Table S3, Supporting Information). The relative configuration of 3 was established by comparing its spectroscopic data with values for the known compound 6β-acetoxysandaracopimaradiene-1α,9αdiol (9)11 and by the analysis of coupling constants and NOE correlations. The small coupling constant at H-1 [d, (J = 1.2 Hz)] also depicted the α-axial orientation of the hydroxy group at C-1. The coupling constants for H-5/H-6 and H-6/H-7 were both revealed to be 2.4 Hz, implying that H-6 is in an αequatorial orientation and the acetoxy group on C-6 is in a βaxial orientation, and the hydroxy group at C-7 was suggested to be α-axially oriented, which was also in agreement with the NOE correlations clearly observed between H-7 and H-14. Therefore, the structure of compound 3 (boesenberol C) was concluded to be 6β-acetoxypimara-8(14),15-diene-1α,7α,9αtriol. Boesenberol D (4) was separated as a colorless, amorphous solid, and its molecular formula was established by HRESIMS as C22H34O4 (MW 362). Compound 4 exhibited a structural similarity to 3 but with the lack of a hydroxy group at C-1. The relative configuration of 4 was assigned by comparing the spectroscopic data with those of the known compound 911 and compound 3 in having C-6β acetoxy and C-7α hydroxy groups, which were also supported by NOE correlations (H-7/H-14). Therefore, the structure of compound 4 (boesenberol D) was revealed as 6β-acetoxypimara-8(14),15-diene-7α,9α-diol. Boesenberol E (5) was isolated as a colorless, amorphous solid, and HRESIMS and 13C NMR data suggested its molecular formula to be C20H32O3 (MW 320). Compounds 5 and 2 have the same molecular formula, but have different positions and configurations of their hydroxy groups. On the basis of the HMBC correlations of H-7 [δH 4.42 (ddd, J = 2.4, 6.0, 12.0 Hz)] to C-8/C-14 and H-11 [δH 4.13 (dd, J = 4.8, 10.2 Hz)] to C-10/C-12/C-13, two secondary hydroxy groups could be located at C-7 and C-11 (Figure S1 and Table S5, Supporting Information). The relative configuration of 5 was assigned by comparing its spectroscopic data with analogous

C NMR (Tables 1 and 2) spectra of 2 showed signals for a pimarane diterpene-type skeleton. The HMBC correlations (Figure S1 and Table S2, Supporting Information) between H1 and C-3/C-20 and between H-7 and C-5/C-9 showed the attachment of two hydroxy groups at C-1 and C-7. HMBC correlations from H3-17 to C-12, C-13, C-14, and C-15, from H-16 to C-13, and from H-12 to C-13, C-14, and C-15 revealed that a methyl group and vinyl group are connected to C-13. These data suggested that 2 is a pimarane diterpene.16 The relative configuration of 2 was assigned by comparing its spectroscopic data with analogous values for the known compound sandaracopimaradien-1α,9α-diol (15).11 The H-1 proton [δH 3.99 (s)] appeared as a singlet, showing a very small coupling constant with H2-2, which indicated the equatorial orientation of H-1, and, thus, the hydroxy group at C-1 was assigned as α-axially oriented, as supported by a comparison with the known compound sandaracopimaradiene-1α,9α-diol (15).11 The H-5 proton was assigned in an axial position depending on its coupling constants (dd, J = 3.0, 13.8 Hz). The axial−axial coupling constant between H-5 and H2-6 was 13.8 Hz, and the axial−equatorial coupling constant was 3.0 Hz. Similarly, small coupling constants were observed between H2-6 and H-7 (J6α,7 = J6β,7 = 3.0 Hz). Therefore, the H-7 proton was assigned as equatorial and the hydroxy group at C-7 as α-axially oriented. A NOE difference experiment revealed that a NOE interaction was observed clearly between H-7 (δH 4.20) and H14 (an olefinic proton at δH 5.58), which also supported the equatorial orientation of H-7. Therefore, the structure of 2 (boesenberol B) was revealed as pimara-8(14),15-diene1α,7α,9α-triol. Boesenberol C (3) was isolated as a colorless, amorphous solid, and its molecular formula C22H34O5 (MW 378) was established by HRESIMS. Compound 3 showed 1H and 13C NMR data (Tables 1 and 2) revealing that it is also a pimarane diterpene with a structural similarity to 2. The HMBC correlations from H-6 (δH 5.38) to C-8 (δC 133.0), C-10 (δC 43.8), and an acetoxy carbonyl (C-21, δC 170.2) suggested that C

DOI: 10.1021/acs.jnatprod.6b00424 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 2. 1H NMR Spectroscopic Data (600 MHz, CDCl3) of Bosenberols A−D (1−4) (δ in ppm and J Values in (Hz) in Parentheses)a

Table 3. 1H NMR Spectroscopic Data (600 MHz, CDCl3) of Bosenberols E−H (5−8) (δ in ppm and J Values in (Hz) in Parentheses)a

position

position

1

2

3

1α 1β 2α

5α

3.81, t (1.8) 2.04, ddt (13.8, 4.2, 1.8) 1.53, dq (13.8, 3.0) 1.68, td (13.8, 4.2) 1.11, dt (13.8, 3.0) 1.62, br s

6α

4.55, br d (4.8)

2β 3α 3β

6β 7α

11α

2.27, dt (18.0, 4.8) 2.50, dd (18.0, 1.2) 2.66, m

11β

2.45, m

12α

1.97, dt (13.2, 4.2) 1.83, td (13.2, 4.2)

7β

12β 14 15 16a 16b 17 18 19 20 21

5.90, dd (17.4, 10.8) 4.94, d (17.4) 5.04, d (10.8) 1.15, s 1.23, s 1.01, s 1.43, s

4 1.77, td (13.2, 3.6) 1.37,b m 1.27, dd (13.2, 3.6) 1.37,b m

1α 1β 2α

1.49, m 1.49,b m 1.49,b m

2β

1.49,b m

3α 3β

1.49,b m 1.38, m

5α

1.90, dd (12.0, 2.4) 1.99, ddd (12.0, 6.0, 2.4) 1.26, q (12.0) 4.42, ddd (12.0, 6.0, 2.4)

3.99, br s 1.52, qd (13.8, 3.0) 1.76,b m

4.02, d (1.2) 1.50, m

1.15, dt (13.8, 3.0) 1.85, td (13.8, 3.0) 2.43, dd (13.8, 3.0) 1.62, td (13.8, 3.0) 1.85, dt (13.8, 3.0)

1.14, dt (12.6, 2.4) 1.93, td (12.6, 3.0) 2.59, d (2.4)

1.57, dt (13.2, 3.6) 1.50, m 2.14, d (2.4)

6α

5.38, t (2.4)

5.29, t (2.4)

6β 7α

4.20, t (3.0) b

1.76, m 1.95, td (13.8, 3.0) 1.43, m 1.81, dd (13.8, 3.0) 5.58, d (1.2) 5.82, dd (17.7, 10.5) 5.00, d (17.7) 4.96, d (10.5) 1.01, s 0.90, s 0.98, s 0.79, s

1.88, m

5 b

3.89, d (2.4)

1.76, dt (13.8, 3.0) 2.06, td (13.8, 3.0) 1.44, m

1.27, dd (13.2, 3.6) 1.50, m

1.82, dd (13.8, 3.0) 5.66, s 5.82, dd (17.4, 10.8) 5.03, d (17.4) 4.97, d (10.8) 1.05, s 1.02, s 1.07, s 1.07, s 1.99, s

11β 12α 12β 14α 14β 15

1.71, td (13.2, 3.6) 1.71, td (13.2, 3.6) 5.66, s 5.81, dd (17.7, 10.8) 5.00, d (17.7) 4.97, d (10.8) 1.05, s 1.01, s 1.00, s 1.15, s 2.00, s

16a 16b 17 18 19 20 21 22

7 b

8 b

1.68, m 1.61,b m 1.48, dt (13.8, 3.0) 1.68,b m

1.65, m 1.65,b m 1.65,b m

1.63, m 1.63,b m 1.63,b m

1.50, m

1.39,b m 1.17, dd (13.8, 3.0) 1.27, br s

1.36,b m 1.36,b m 1.56, br s

1.46, dt (13.8, 3.6) 1.38,b m 1.17, dd (13.8, 3.6) 1.31, br s

4.27, br s

4.39, br s

4.38, br s

3.96, br s 2.01, m

3.26, br s 2.20,b m

4.13, dd (10.2, 4.8) 1.63,b m 1.63,b m

1.95, m

1.99, m

3.46, br s 1.94, dd (10.8, 6.0) 1.98, m

1.61,b m 1.39,b m

2.20,b m 1.28, m

5.73, br s

3.48, s

5.86, dd (17.4, 10.8) 5.02, d (17.4) 4.96, d (10.8) 1.08, s 0.86, s 0.93, s 0.91, s

5.72, dd (17.4, 11.1) 5.05, d (17.4) 4.97, d (11.1) 1.10, s 1.21, s 0.99, s 1.32, s 3.54, s

7β 11α 3.85, d (2.4)

6 b

2.98, s 6.06, dd (17.4, 10.8) 5.03, d (17.4) 5.00, d (10.8) 0.86, s 1.21, s 0.98, s 1.32, s 3.45, s 3.39, s

1.38,b m 1.56, ddd (10.8, 6.0, 3.0) 3.40, s 5.71, dd (17.4, 10.8) 5.04, d (17.4) 4.95, d (10.8) 1.09, s 1.20, s 0.99, s 1.30, s 3.44, s 3.51, s

δ values were measured from the HMQC spectrum. bOverlapping resonances within the same column. a

a δ values were measured from the HMQC spectrum. bOverlapping resonances within the same column.

Hz, H-15), 5.05 (d, J = 17.4 Hz, H-16a), 4.97 (d, J = 11.1 Hz, H-16b)], which supported the structure of this compound being a pimarane diterpene.16 The 13C NMR (Table 1) spectrum showed four olefinic carbons (δC 125.6, 146.9, 143.9, and 112.7), four methines including three oxygenated signals (δC 49.1, 71.8, 71.6, and 81.8), five methylenes (δC 39.1, 19.0, 42.8, 21.3, and 29.7), three quaternary carbons (δC 33.7, 38.1, and 40.0), and five methyls including an oxygenated signal (δC 23.5, 24.1, 33.4, 21.8, and 62.0). The HMBC correlations from H-6 (δH 4.27) to C-5, C-8, and C-10, from H-7 (δH 3.96) to C5, C-8, C-9, and C-14, and from H-14 (δH 3.48) to C-9, C-15, C-17, and the methoxy carbon (C-21) were used to assign the positions of the hydroxy groups at C-6 and C-7 and the methoxy group at C-14 (Figure S1 and Table S6, Supporting Information). The relative configuration of 6 was determined by comparing its spectroscopic data with those of the known compound curcumrinol A.18 The small coupling constants of H-6 (δH 4.27, br s) and H-7 (δH 3.96, br s) suggested that the hydroxy groups at C-6 and C-7 are β-axial and α-axial, respectively. In addition, the equatorial H-7 proton showed NOE correlations with the equatorial methoxy protons at C-14, while the axial H-14 proton showed an NOE correlation with

data of the known compound acerifolin B.17 The large coupling constant of H-11 [δH 4.13 (dd, J = 4.8, 10.2 Hz)] also supported the α-equatorial orientation of the hydroxy group at C-11. The H-5 proton was α-axially oriented from its coupling constant [δH 1.90 (dd, J = 2.4, 12.0 Hz)]. The coupling constant between H-5 and H-6β (δH 1.26) was 12 Hz, suggesting the axial−axial orientation of these protons, while the J value between H-6β and H-7 (δH 4.42) was also 12.0 Hz, suggesting that H-7 was α-axially oriented and the hydroxy group at C-7 was β-equatorially oriented. In addition, H-5 and H-7 showed a NOE correlation that also supported the βequatorial orientation of the hydroxy group at C-7. Hence, the structure of 5 (boesenberol E) was proposed as pimara8(14),15-diene-7β,9α,11α-triol. Boesenberol F (6) was obtained as a colorless, amorphous solid, and its molecular formula was assigned as C21H34O3 (MW 334) on the basis of HRESIMS. The 1H NMR (Table 3) spectrum displayed singlets for four tertiary methyl groups [δH 1.10 (H3-17), 1.21 (H3-18), 0.99 (H3-19), and 1.32 (H3-20)] and a monosubstituted vinyl group [δH 5.72 (dd, J = 11.1, 17.4 D

DOI: 10.1021/acs.jnatprod.6b00424 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Effects of compound 9 on TRAIL-resistant human gastric adenocarcinoma (AGS) and human embryonic kidney (HEK293) cells. Effects of compound 9, luteolin (positive control: Lut), and DMSO (negative control: Cont.) in the presence and absence of TRAIL. 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 compound 9 and TRAIL (100 ng/mL) for 24 h. Viability was evaluated by the FMCA method. Data represent the means ± SD (n = 4). Significant differences in cell viability between the presence and absence of TRAIL were observed for AGS cells, while there were no significant differences for HEK293 cells.

whether it exerted any cytotoxicity against noncancerous cells, the effects of the combined treatment of TRAIL (100 ng/mL) and 9 (10, 20, 30, 40, and 50 μM) on human embryonic kidney (HEK293) cells were examined, and the results obtained revealed no significant effects on viability at 10, 20, and 30 μM (Figure 1). Thus, compound 9 was concluded to selectively kill malignant cells without affecting the noncancerous HEK293 cells up to 30 μM. In order to elucidate the intracellular apoptotic mechanism of 9 against AGS cells, the levels of TRAIL-induced apoptoticrelated proteins were investigated using Western blotting analysis. The synergistic induction of apoptosis by the combined treatment of 9 and TRAIL was assessed in AGS cells, and the treatment of these cells with 9 induced DR4 and DR5 protein levels after 24 h in a dose-dependent manner (Figure 2). TRAIL-induced apoptosis is initiated after binding of TRAIL to DR4 and DR5 receptors, and signals are transmitted to cells through the functional cytoplasmic Fas associated death domain (trimerization of Fas and pro-caspase8), which leads to the transformation of procaspase-8 into caspase-8.4 Figure 2 shows that 9 also enhanced Fas levels in a

equatorial H-15, thereby suggesting that the methoxy group at C-14 is β-equatorially oriented. Therefore, the structure of compound 6 (boesenberol F) was proposed as 14βmethoxypimara-8,15-diene-6β,7α-diol. Boesenberol G (7) was isolated as a colorless, amorphous solid, and the HRESIMS and 13C NMR data were used to determine its molecular formula as C22H36O3 (MW 348). Compounds 6 and 7 showed structural similarities, except for the absence of a hydroxy group and the presence of a methoxy group at C-7 in 7, which was consistent with its HMBC correlations between the oxygenated methine (δH 3.26, br s, H7) and C-5, C-8, C-9, and the methoxy carbon (C-21) (Figure S1 and Table S7, Supporting Information). The relative configuration of 7 was assigned by comparing its spectroscopic data with the known compound kaempulchraol A.12 The H-5 and H-6 resonances both appeared as broad singlets, indicating only a small coupling constant between them; thus H-6 was suggested to have an α-equatorial configuration. Furthermore, unlike compound 6, compound 7 displayed a substantial NOE correlation from H-7 to H-14, but not MeO-14, implying that H-7 and H-14 are both β-equatorial. Therefore, the structure of 7 (boesenberol G) was concluded to be 7α,14α-dimethoxypimara-8,15-dien-6β-ol. Boesenberol H (8) was obtained as a colorless, amorphous solid, and its molecular formula was shown to be C22H36O3 (MW 348) by HRESIMS. The 1H and 13C NMR (Tables 1 and 3) spectroscopic data of 8 were almost identical to those of 7, except for the β-orientation of the methoxy group on C-14 (δH 3.40, s), which was confirmed by the NOE correlations observed from OMe-14 to H3-17 (δH 1.09, s) and from H-14 to H-15. Therefore, the structure of 8 (boesenberol H) was revealed as 7α,14β-dimethoxypimara-8,15-dien-6β-ol. The isolated compounds 1−18 exhibited TRAIL-resistanceovercoming activity in TRAIL-resistant AGS cells, as shown in Figure S3, Supporting Information. Among them, compound 9 (6β-acetoxysandaracopimaradiene-1α,9α-diol) exhibited potent TRAIL-resistance-overcoming activity (Figure 1) and was selected for further studies using AGS cells because a significant quantity of this compound was obtained. In order to clarify

Figure 2. Western blot analysis of DR4, DR5, p53, Fas, and CHOP protein levels in AGS cells after 24 h treatment with 9. The AGS cells were seeded in a culture plate for 24 h, treated with the indicated concentrations of 9 for 24 h, and then analyzed by Western blotting. βActin was used as internal control. E

DOI: 10.1021/acs.jnatprod.6b00424 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Caspase-8 is a critical mediator of death-receptor-induced apoptosis, and GSK-3β suppresses the activity of caspase-8. GSK-3β-inhibitor-induced TRAIL sensitization is known to depend on the activity of caspase-8.22 Western blotting results showed that 9 significantly triggered the down-regulation of the cell survival proteins GSK-3β and Bcl-2 in a dose-dependent manner (Figure 3). c-FLIP (cellular FLICE-like inhibitory protein) is an antagonist of caspases-8 and -10, preventing the binding of these caspases to DISC and, thus, inhibiting the autolytic cleavage and subsequent activation of caspase-8.7 c-FLIP is expressed in various tumor cells and its expression is associated with enhanced tumorigenicity and poor clinical outcomes in many types of cancers due to TRAIL resistance. In the present study, treatment with compound 9 significantly down-regulated c-FLIP levels in AGS cells (Figure 4). Therefore, subtoxic doses of 9 appear to have sensitized human AGS cells to TRAILinduced apoptosis by down-regulating c-FLIP protein levels.

dose-dependent manner. The proteolytic activation of caspase8 further induces two different apoptotic pathways, and cleaved caspase-8 has been found to play a significant role in cell apoptosis.4 Figure 3 shows that compound 9 up-regulated the levels of cleaved caspase-8.

Figure 3. Western blot analysis of GSK-3β, Bcl-2, Bax, and cleaved caspase-3, -8, and -9 protein levels in AGS cells after 24 h treatment with 9. The AGS cells were seeded in a culture plate for 24 h, treated with the indicated concentrations of 9 for 24 h, and then analyzed by Western blotting. β-Actin was used as internal control. Figure 4. Western blot analysis of c-FLIP protein levels in AGS cells after 24 h treatment with 9. The AGS cells were seeded in a culture plate for 24 h, treated with the indicated concentrations of 9 for 24 h, and then analyzed by Western blotting. β-Actin was used as internal control.

Cleaved caspase-8 or proteolytic activated caspase-8 further activates Bid, which, in turn, translocates to mitochondria and activates mitochondrial pathways.4 In mitochondrial pathways, death signals cause changes in mitochondrial outer membrane permeability by releasing the mitochondrial membrane proteins Bax and Bak, which results in the subsequent release of cytochrome c, leading to the formation of an apoptosome with Apaf-1 (apoptotic protease-activating factor 1) and caspase-9.5 The Apaf-1-mediated proteolytic activation of caspase-9 forms activated or cleaved caspase-9.5 The results of the Western blotting analysis showed that 9 up-regulated the levels of Bax and cleaved caspase-9 (Figure 3). Cleaved caspase-9 then activates caspase 3/7. Caspase-3 is one of the key executioners of apoptosis, and the proteolytic activation of caspase-3 is the final stage of apoptosis.5 The treatment of AGS cells with 9 for 24 h increased the levels of cleaved caspase-3 (Figure 3). The present study exhibited that compound 9 enhanced the levels of cleaved caspases-8, -9, and -3 (Figure 3). Since the proteolytic activation of caspase-3 is the final stage of apoptosis, 9 potentiated TRAIL to induce apoptosis. The important tumor suppressor protein p53 acts as a transcriptional factor of the proteins involved in the TRAIL signal.19 This up-regulates the expression of Apaf 1 as well as the TRAIL receptors DR4 and DR5 and pro-apoptotic mitochondrial membrane proteins Bax and Bak. The p53 protein has also been shown to suppress the expression of certain antiapoptotic genes such as Bcl-2, Bcl-xl, and survivin.4 The p53 protein levels was investigated using Western blot analysis, and the results obtained showed that 9 enhanced the p53 protein levels in a dose-dependent manner in TRAILresistant AGS cells (Figure 2). A previous study reported that CHOP enhanced the level of DR5 for TRAIL-induced apoptosis.20 CHOP has been identified as an endoplasmic reticulum (ER) stress marker protein involved in ER stressmediated apoptosis.21 In the present study, compound 9 was shown to up-regulate CHOP protein levels in a dose-dependent manner (Figure 2).

In order to rationalize the relationship between TRAIL and 9 in the TRAIL-inducing apoptosis mechanism, the levels of cleaved caspases-8, -9, and -3 were investigated using 9 and TRAIL together by Western blotting analysis. The results showed that the combined treatment of TRAIL (100 ng/mL) and 9 at 16 μM in AGS cells for 24 h markedly increased the levels of cleaved caspases-8, -9, and -3 more than 9 or TRAIL alone. The results of the Western blotting band intensity value analysis exhibited that 9 potentiated the activity of cleaved caspases-8, -9, and -3 by 65%, 75%, and 68%, respectively, with TRAIL (Figure 5). Thus, it was concluded that 9 mediated the levels of cleaved caspases-8, -9, and -3, while this compound markedly enhanced the levels of these caspases in the presence

Figure 5. Western blot analysis of a combined effect of 9 and TRAIL on cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 protein levels in AGS cells. The AGS cells were seeded in a culture plate for 24 h, treated with the indicated concentration of 9, TRAIL, and 9 and TRAIL together for 24 h, and then analyzed by Western blotting. The value under the band indicates the band intensity (%). β-Actin was used as internal control. F

DOI: 10.1021/acs.jnatprod.6b00424 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

28 cm) and eluted with a MeOH/H2O (5:5 to 10:0) solvent system to afford fractions 3A to 3L and compounds 1 (3.8 mg) and 10 (25 mg). Fraction 2D (51 mg) was eluted with preparative ODS HPLC [YMC Pack Pro C18 (⦶ 10 i.d. × 250 mm), flow rate: 2 mL/min, RI and UV detection at 254 nm] using 80% MeOH to afford compounds 2 (8 mg), 9 (16 mg), and 10 (1.3 mg). Fraction 3J (9 mg) was subjected to preparative ODS HPLC [YMC Pack Pro C18, ⦶ 10 i.d. × 250 mm, flow rate: 2 mL/min, RI and UV detection at 254 nm] and eluted with 85% MeOH to afford compounds 11 (1.5 mg) and 12 (1.2 mg). Fraction 3I (11 mg) was eluted with preparative ODS HPLC [YMC Pack Pro C18 (φ 10 i.d. × 250 mm), flow rate: 2 mL/min, RI and UV detection at 254 nm] using 85% MeOH to afford compounds 2 (1.5 mg) and 11 (3.5 mg). Fraction 3H (78 mg) was subjected to preparative ODS HPLC [YMC Pack Pro C18, (φ 10 i.d. × 250 mm), flow rate: 2 mL/min, RI and UV detection at 254 nm], and 75% MeOH was used to afford fractions 7A to 7L and compounds 2 (3.5 mg), 3 (8 mg), 9 (4 mg), and 10 (8 mg). Fraction 1J (69 mg) was separated using preparative ODS HPLC [YMC Pack Pro C18 (⦶ 10 i.d. × 250 mm), flow rate: 2 mL/min, RI and UV detection at 254 nm] and eluted with 80% MeOH to afford compounds 4 (2 mg), 13 (3.5 mg), 14 (2 mg), 15 (4 mg), and 17 (2 mg). Fraction 1O (59 mg) was purified using preparative ODS HPLC [YMC Pack Pro C18 (⦶ 10 i.d. × 250 mm), flow rate: 2 mL/min, RI and UV detection at 254 nm], with 70% MeOH as eluent to afford compounds 5 (1.2 mg), 16 (2 mg), and 18 (0.9 mg). Fraction 7H (22 mg) was separated using preparative ODS HPLC [Inertsil ODS-3 (⦶ 10 i.d. × 250 mm), flow rate: 2 mL/min, RI and UV detection at 254 nm], eluted with 85% MeOH, to afford compounds 6 (2.5 mg), 7 (1.5 mg), and 8 (5.5 mg). Boesenberol A (1): colorless, amorphous solid; [α]25D +92.8 (c 0.3, CHCl3); IR (ATR) νmax 3470, 2924, 1651 cm−1; 1H and 13C NMR data, see Tables 1 and 2, and 2D NMR data, see Table S1, Supporting Information; HRESIMS m/z 659.4289 [2M + Na]+ (Δ +0.1 mmu), calcd for C40H60O6Na, 659.4288. Boesenberol B (2): colorless, amorphous solid; [α]25D −16.4 (c 0.3, CHCl3); IR (ATR) νmax 3387, 3318, 2939 cm−1; 1H and 13C NMR data, see Tables 1 and 2, and 2D NMR data, see Table S2, Supporting Information; HRESIMS m/z 343.2255 [M + Na]+ (Δ +0.6 mmu), calcd for C20H32O3Na, 343.2249. Boesenberol C (3): colorless, amorphous solid; [α]25D −33.5 (c 0.3, CHCl3); IR (ATR) νmax 3362, 2923, 1733, 1242 cm−1; 1H and 13C NMR data, see Tables 1 and 2, and 2D NMR data, see Table S3, Supporting Information; HRESIMS m/z 779.4707 [2M + Na]+ (Δ −0.3 mmu), calcd for C44H68O10Na, 779.4710. Boesenberol D (4): colorless, amorphous solid; [α]25D +23.4 (c 0.3, CHCl3); IR (ATR) νmax 3424, 2928, 1733, 1372, 1243 cm−1; 1H and 13 C NMR data, see Tables 1 and 2, and 2D NMR data, see Table S4, Supporting Information; HRESIMS m/z 385.2318 [M + Na]+ (Δ −3.7 mmu), calcd for C22H34O4Na, 385.2355. Boesenberol E (5): colorless, amorphous solid; [α]25D +19.0 (c 0.3, CHCl3); IR (ATR) νmax 3853, 3418, 2944, 1456 cm−1; 1H and 13C NMR data, see Tables 1 and 3, and 2D NMR data, see Table S5, Supporting Information; HRESIMS m/z 663.4562 [2M + Na]+ (Δ −3.9 mmu), calcd for C40H64O6Na, 663.4601. Boesenberol F (6): colorless, amorphous solid; [α]25D +58.1 (c 0.3, CHCl3); IR (ATR) νmax 3419, 2931, 1737, 1456 cm−1; 1H and 13C NMR data, see Tables 1 and 3, and 2D NMR data, see Table S6, Supporting Information; HRESIMS m/z 357.2359 [M + Na]+ (Δ −4.6 mmu), calcd for C21H34O3Na, 357.2406. Boesenberol G (7): colorless, amorphous solid; [α]25D −18.7 (c 0.3, CHCl3); IR (ATR) νmax 3477, 2930, 1737, 1458 cm−1; 1H and 13C NMR data, see Tables 1 and 3, and 2D NMR data, see Table S7, Supporting Information; HRESIMS m/z 371.2565 [M + Na]+ (Δ +0.3 mmu), calcd for C22H36O3Na, 371.2562. Boesenberol H (8): colorless, amorphous solid; [α]25D +64.9 (c 0.5, CHCl3); IR (ATR) νmax 3435, 2927, 1460, 1083 cm−1; 1H and 13C NMR data, see Tables 1 and 3, and 2D NMR data, see Table S8, Supporting Information; HRESIMS m/z 371.2572 [M + Na]+ (Δ +1.0 mmu), calcd for C22H36O3Na, 371.2562. Cell Cultures. AGS and HEK293 cells were purchased from the ATCC. AGS cells were cultured in Roswell Park Memorial Institute

of TRAIL. Since the proteolytic activation of caspases-8, -9, and -3 plays a significant role in TRAIL-mediated apoptosis, 9 influences the TRAIL to induce apoptosis in AGS cells. Previous studies have reported that dibenzylideneacetone,23 ginsenoside 20(S)-Rg3,24 and luteolin25 increase the levels of cleaved caspase-8, cleaved caspase-9, and cleaved caspase-3 in HCT116 and AGS cells. Another study reported that bortezomib enhanced the level of cleaved caspase-3 in RPMI 8226 and KMS-11 cells (bone marrow mononuclear cells).26 It was previously demonstrated that parviflorene F enhanced the levels of cleaved caspase-8 in HeLa cells.27 In the present study, the activity-guided separation of B. pandurata resulted in the isolation of 17 pimarane diterpenes and one monoterpene compound. Among these, compounds 1−8 are new pimarane diterpenes. All compounds exhibited TRAIL-resistance-overcoming activity in TRAIL-resistant AGS cells. Western blotting analysis results showed that 9 enhanced the levels of the apoptosis-inducing proteins DR4, DR5, cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, Fas, p53, CHOP, and the mitochondrial membrane protein Bax, but down-regulated the cell survival proteins Bcl-2, GSK-3β, and cFLIP in a dose-dependent manner. In the presence of TRAIL, 9 potentiated the levels of cleaved caspases-8, -9, and -3 in TRAIL-resistant AGS cells. In addition, compound 9 did not decrease the viability of noncancerous cells at concentrations up to 30 μM. To the best of our knowledge, this is the first report of the TRAIL-resistance-overcoming activity of 6βacetoxysandaracopimaradiene-1α,9α-diol (9) in TRAIL-resistant AGS cells.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO P-1020 polarimeter. ATR-IR spectra were measured on a JASCO FT-IR 230 spectrophotometer. NMR spectra were recorded on JEOL ECZ600 and ECA600 NMR spectrometers with a deuterated solvent, of which the chemical shifts were used as an internal standard. HRESIMS were obtained on a JEOL JMS-T100LP mass spectrometer. Column chromatography was performed using silica gel 60N (Fuji Silysia Chemical Co., Kasugai, Japan) and Chromatorex ODS (Fuji Silysia Chemical Co.). Preparative HPLC was performed using YMC-Pack ODS-AM (YMC Co. Ltd., Kyoto, Japan) and Inertsil ODS-3 (GL Sciences Inc., Japan). Protein concentrations were measured with a Nano Drop 2000 spectrophotometer (Thermo), and Western blotting membranes were analyzed by a Bio-Rad (ChemiDoc XRS+) instrument. Fluorescence was measured using a Fluoroskan Ascent instrument (Thermo Fisher Scientific, Waltham, MA, USA). Plant Material. The rhizomes of B. pandurata were collected from Khon Kaen, Thailand, during 2008−2011, and identified by one of the authors (T. Kowithayakorn). A voucher specimen (KKP87) was deposited at the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Japan. Extraction and Isolation. The air-dried and ground rhizomes of B. pandurata (150 g) were subjected to extraction with MeOH at room temperature for 5 days, followed by homogenization and filtration and then evaporation and vacuum desiccation overnight to obtain a crude extract (18.5 g). The extract was partitioned between nhexane, EtOAc, and n-BuOH (400 mL × 3) to obtain dried n-hexane (10.0 g), EtOAc (3.0 g), n-BuOH (2.1 g), and water (2.23 g) fractions. The EtOAc fraction (3.0 g) was subjected to silica gel 60N column chromatography (⦶ 5 × 30 cm) and eluted with a hexane/EtOAc solvent system (10:0−0:1) to afford fractions 1A to 1T and compound 9 (83 mg). Fraction 1 M (133 mg) was eluted with ODS flash column chromatography (⦶ 1.8 × 25 cm) using MeOH/H2O (6:4 to 10:0) to afford fractions 2A to 2F and compound 10 (25 mg). Fraction 1N (247 g) was subjected to ODS flash column chromatography (⦶ 1.8 × G

DOI: 10.1021/acs.jnatprod.6b00424 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

■

(RPMI)-1640 medium (Wako, Osaka, Japan) with fetal bovine serum (10% FBS) and 1% penicillin−streptomycin (Sigma-Aldrich, St. Louis, MO, USA). HEK293 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Wako) with 10% FBS and 1% penicillin− streptomycin. Cultures were maintained in a humidifier incubator at 37 °C in 5% CO2/95% air. Viability Assay (FMCA). Cell viability was assessed using a fluorometric microculture cytotoxicity assay (FMCA) in the presence and absence of TRAIL using TRAIL-resistant AGS cells and HEK293 cells. Cells were seeded on 96-well culture plates (6 × 103 cells per well) in 200 μL of medium containing 10% FBS. Then these were 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 period, the cells were washed with PBS, and 200 μL of PBS containing fluorescein (10 μg/mL)8 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 a Fluoroskan Ascent apparatus. Luteolin (purity ≥98%, Sigma-Aldrich) was used as a positive control. 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 (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% skimmed milk in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 1 h, the membrane was incubated at room temperature with primary antibodies [anti-p53 (1:4000, #P5813, Sigma-Aldrich); anti-DR5 (1:500, Sigma-Aldrich); anti-DR4 (1:500, Sigma-Aldrich); anti-Fas (1:1000, Sigma-Aldrich); anti-c-FLIP (1:1000, Alexis); anti-GSK-3β (1:1000, Santa Cruz Biotechnology, CA, USA); anti-CHOP (1:1000, #WH0001649M1-100UG, SigmaAldrich); anti-Bax (1:1000, #H1505, Santa Cruz Biotechnology); antiBcl-2 (1:1000, #117 K4812, Sigma-Aldrich); anti-cleaved caspase-3 (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-cleaved caspase-8 (1:1000, #Q14790, Cell Signaling Technology); anti-cleaved caspase-9 (1:1000, #P55211, Cell Signaling Technology); and anti-βactin (1:4000, #A2228, Sigma-Aldrich)] at room temperature overnight. 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) at room temperature for 1 h. 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).

■

ACKNOWLEDGMENTS This study was supported by KAKENHI Grant Number 23102008 from MEXT, 26305001 and 26293022 from JSPS, the Uehara Memorial Foundation, and the Tokyo Biochemical Research Foundation (TBRF).

■

REFERENCES

(1) Trivedi, R.; Mishra, D. P. Front. Oncol. 2015, 5, 1−20. (2) Hellwig, C. T.; Rehm, M. Mol. Cancer Ther. 2012, 11, 3−13. (3) Ahmed, F.; Ishibashi, M. Chem. Pharm. Bull. 2016, 64, 119−127. (4) Wu, G. S. Cancer Lett. 2009, 285, 1−5. (5) Amarante-Mendes, G. P.; Griffith, T. S. Pharmacol. Ther. 2015, 155, 117−131. (6) Yang, J. F.; Cao, J. G.; Tian, L.; Liu, F. Cancer Chemother. Pharmacol. 2012, 69, 195−206. (7) Piggott, L.; Omidvar, N.; Perez, S. M.; Eberl, M.; Clarkson, R. W. Breast Cancer Res. 2011, 13, 2−15. (8) Lindhagen, E.; Nygren, P.; Larsson, R. Nat. Protoc. 2008, 3, 1364−1369. (9) Chahyadi, A.; Hartati, R.; Wirasutisna, K. R.; Elfahmi. Procedia Chem. 2014, 13, 13−37. (10) Elfahmi; Woerdenberg, H. J.; Kayse, O. J. Herb. Med. 2014, 4, 51−73. (11) Prawat, U.; Tuntiwachwuttikul, P.; Taylor, W. C.; Engelhard, T.; Skelton, B. W.; White, A. H. Phytochemistry 1993, 32, 991−997. (12) Win, N. N.; Ito, T.; Aimaiti, S.; Imagawa, H.; Ngwe, H.; Abe, I.; Morita, H. J. Nat. Prod. 2015, 78, 1113−1118. (13) Tuchinda, P.; Udchachon, J.; Reutrakul, V.; Santisuk, T.; Skelton, B. W.; White, A. H.; Taylor, W. C. Phytochemistry 1994, 36, 731−734. (14) Thongnest, S.; Mahidol, C.; Sutthivaiyakit, S.; Ruchirawat, S. J. Nat. Prod. 2005, 68, 1632−1636. (15) Liu, X.; Wu, Q.-X.; Shi, Y.-P. J. Chin. Chem. Soc. 2005, 52, 369− 374. (16) Meragelman, T. L.; Silva, G. L.; Mongelli, E.; Gil, R. R. Phytochemistry 2003, 62, 569−572. (17) Wu, X.-D.; Zhang, L.-C.; He, J.; Li, G.-T.; Ding, L.-F.; Gao, X.; Dong, L.-B.; Song, L.-D.; Li, Y.; Zhao, Q.-S. J. Asian Nat. Prod. Res. 2013, 15, 151−157. (18) Huang, W.; Zhang, P.; Jin, Y.-C.; Shi, Q.; Cheng, Y.-Y.; Qu, H.B.; Ma, Z.-J. Helv. Chim. Acta 2008, 91, 944−950. (19) Zhao, J.; Lu, Y.; Shen, H. M. Cancer Lett. 2012, 314, 8−23. (20) Gupta, S. C.; Sajin, F. K.; Nair, M. S.; Mo, Y. Y.; Aggarwal, B. B. J. Biol. Chem. 2013, 288, 32343−32356. (21) Yang, J. F.; Cao, J. G.; Tian, L.; Liu, F. Cancer Chemother. Pharmacol. 2012, 69, 195−206. (22) Liabo, X.; Zhang, L.; Thrasher, B. Mol. Cancer Ther. 2003, 2, 1215−1222. (23) Prasad, S.; Yadav, V. R.; Ravindran, J.; Aggarwal, B. B. Cancer Res. 2011, 71, 538−549. (24) Park, E. H.; Kim, Y. J.; Yamabe, N.; Park, S. H.; Kim, H. K.; Jang, H. J.; Kim, J. H.; Cheon, G. J.; Ham, J.; Kang, K. S. J. Ginseng Res. 2014, 38, 22−27. (25) Wu, B.; Zhang, Q.; Shen, W.; Zhu, J. Mol. Cell. Biochem. 2008, 313, 125−132. (26) Miki, H.; Nakamura, S.; Oda, A.; Amachi, R.; Watanabe, K.; Hanson, D.; Teramachi, J.; Hiasa, M.; Yagi, H.; Sogabe, K.; Takahashi, M.; Maruhashi, T.; Udaka, K.; Harada, T.; Fuji, S.; Nakano, A.; Ri, K. M.; Iida, S.; Ozaki, S.; Matsumoto, T.; Abe, M. Int. J. Myelom. 2015, 5, 1−7. (27) Ohtsuki, T.; Tamaki, M.; Toume, K.; Ishibashi, M. Bioorg. Med. Chem. 2008, 16, 1756−1763.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00424.

■

Article

NMR data tables and spectra, structures, and bioactivity results of isolated compounds (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel and fax: +81-43-226-2923. E-mail: [email protected] (M. Ishibashi). Notes

The authors declare no competing financial interest. H

DOI: 10.1021/acs.jnatprod.6b00424 J. Nat. Prod. XXXX, XXX, XXX−XXX