Evaluation of Lignans from Heliopsis helianthoides var. scabra for

Article pubs.acs.org/jnp

Evaluation of Lignans from Heliopsis helianthoides var. scabra for Their Potential Antimetastatic Effects in the Brain Zsanett Hajdu,†,⊥ János Haskó,‡,⊥ István A. Krizbai,‡,§ Imola Wilhelm,‡ Nikoletta Jedlinszki,† Csilla Fazakas,‡ Judit Molnár,‡ Peter Forgo,† Judit Hohmann,*,† and Dezső Csupor† †

Department of Pharmacognosy, University of Szeged, H-6720 Szeged, Hungary Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, H-6726 Szeged, Hungary § Institute of Life Sciences, Vasile Goldiş Western University of Arad, Arad 310414, Romania ‡

S Supporting Information *

ABSTRACT: Two new arylbenzofuran-type neolignans, 1″dehydroegonol 3″-methyl ether (1) and egonol 3″-methyl ether (2), and four known lignan derivatives, namely, helioxanthin (3), (7E)-7,8-dehydroheliobuphthalmin (4), heliobuphthalmin (5), and 7-acetoxyhinokinin (6), were isolated from a chloroform-soluble partition of the methanol extract of the fresh roots of Heliopsis helianthoides var. scabra. These six compounds were evaluated in vitro in terms of their ability to inhibit the various steps involved in brain tumor metastasis formation. Compounds 3 and 4 inhibited the migration of both melanoma and brain endothelial cells, and 3 also reduced the adhesion of melanoma cells to the brain endothelium. Furthermore, 3 and 4 additionally enhanced the barrier function of the blood-brain barrier and the expression of the tight junction protein ZO-1 at the junctions of the endothelial cells. These findings suggest that 3 and 4 may have the potential to interfere with different steps of brain metastasis formation and to enhance the barrier function of cerebral endothelial cells.

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thinwere isolated earlier from the roots of H. helianthoides var. scabra.10,11 Experimental evidence has demonstrated clear antineoplastic effects of enterolignans,12 flax seed,12 or pure lignans such as hydroxymatairesinol2 or honokiol13,14 in animals. A number of investigations have revealed the cytostatic effects of numerous lignans on different types of cancer cell lines.15−18 Certain compounds, including schizandrin B,16 picropodophyllin,17 and podophyllotoxin and their derivatives,19 display marked cytotoxic, antineoplastic, and antimetastatic activities. However, the effects of lignans on the transmigration of metastatic cells through endothelial barriers (a critical step in metastasis formation) have not been investigated so far. Among various types of tumors, melanoma has the highest propensity to form brain metastases: autopsy data indicate brain metastases in approximately 75% of patients who die from metastatic melanoma.20 The intravasation and dissemination of cancer cells through the circulation, adhesion to the endothelium, extravasation, and eventually tumor growth in a remote organ are the elementary steps in the metastatic process.21 In the case of the brain, a barrier system is situated between the circulation and the central nervous system (CNS),

he lignans are a large group of phenolic compounds present in many edible plants, e.g., oil seeds, seaweed, whole grains, fruits, and vegetables.1 Some of the edible plant lignans, such as secoisolariciresinol and matairesinol isolated from flaxseed1 or hydroxymatairesinol from Picea abies,2 are converted by the intestinal bacteria to enterolignans, enterodiol, and enterolactone, of which the latter is thought to be one of the major biologically active lignans in human. The genus Heliopsis comprises 15 species, of which all are restricted to the Americas.3,4 The genus is known worldwide for its ornamental plants, and recognized phytochemically for the production of N-alkylamides. Heliopsis helianthoides var. scabra (Dunal) Fernald (Asteraceae) has been used to relieve lung troubles and strengthen the limbs by North American native peoples.5 Besides N-alkylamides, this species contains lignans as compounds characteristic of the genus. In the genus Heliopsis, different types of lignans have been identified: the dibenzylbutane derivatives, heliobuphthalmin, 8-hydroxyheliobuphthalmin, 7Z-7,8-dehydroheliobuphthalmin, and 2-[(2H-1,3-benzodioxol-5-yl)methyl]-3-[(3,4-dimethoxyphenyl)methyl]succinic acid dimethyl ester; the dibenzylbutyrolactones, heliobuphthalmin lactone and 5,7′-dehydroheliobuphthalmin lactone in the aerial parts of H. buphthalmoides;6−8 and the dibenzylbutyrolactones, hinokinin and 2′-hydroxyhinokinin from the stems of H. longipes.9 Two lignan derivativesthe dibenzylbutyrolactone, helianthoidin, and the arylnaphthalene, helioxan© XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 20, 2014

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dx.doi.org/10.1021/np500508y | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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formed by the blood-brain barrier (BBB) and the bloodcerebrospinal fluid barrier. Since the brain lacks a lymphatic system, metastatic cells have to cross one of these barriers in order to enter the brain. The BBB is an active barrier that restricts the free movement of different solutes and cells between the circulation and the brain. The mechanisms of cellular transmigration have mainly been investigated through the use of immune cells, and several key steps of transmigration have been defined: these include cell arrest, firm adhesion, and transmigration. Unfortunately, much less is known about the transmigration of metastatic cells, though some of its steps may be similar to the transmigration of leukocytes. Adhesion molecules, proteolytic enzymes, and signaling pathways presumably play roles in this process.21 Various lignans have been demonstrated to downregulate different matrix metalloproteinases14,16,22 and cellular adhesion molecules,23−26 such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which are considered to be important in the extravasation of tumor cells. Moreover, treatment with different lignans reduces the migratory potential of highly metastatic tumor cells.22,27 Considering that the biological activities of lignans isolated from the genus Heliopsis have not been examined previously, and as a result of the promising antimetastatic potential of several lignans, the aims of the present study were to isolate and characterize the structures of lignans from H. helianthoides var. scabra and to evaluate their effects on melanoma brain metastasis formation via in vitro models.

performed by means of NOESY experiments. Six lignans (1− 6) from the fresh roots of H. helianthoides var. scabra were identified; two of them are the new arylbenzofuran-type norneolignans, 1″-dehydroegonol 3″-methyl ether (1) and egonol 3″-methyl ether (2). Helioxanthin (3), an arylnaphthalene derivative, was identified earlier from this species.10 The dibenzylbutane dehydroheliobuphthalmin (4) and heliobuphthalmin (5) were isolated for the first time from H. helianthoides var. scabra, although they have been identified earlier in H. buphthalmoides.6 The dibenzylbutyrolactone, 7-acetoxyhinokinin (6), had been identified earlier only from Ruta pinnata.28

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RESULTS AND DISCUSSION Compound Structure Elucidation. Chromatographic separation on H. helianthoides var. scabra resulted in the isolation of six pure compounds. The structures of these compounds were established by mass spectrometry and NMR methods, including 1H NMR, JMOD, 1H−1H-COSY, HMQC, and HMBC experiments, and comparison with literature data. Stereochemical studies and conformational analysis were

Table 1. NMR Spectroscopic Data (500 MHz (1H), 125 MHz (13C), CDCl3) for Compounds 1 and 2 (δ in ppm, J in Hz) 1 position 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ OCH3-7 OCH3-3″ -OCH2O-

2

δC 156.7 100.5 111.8 137.8 104.7 144.8 142.8 131.0 124.8 105.5 148.2 148.2 108.6 119.3 133.1 124.8 73.2 56.1 56.9 101.3

δH C CH CH C CH C C C C CH C C CH CH CH CH

δC

6.82 s 7.14 brs 6.87 brs

7.32 d (0.7)

6.87 7.40 6.67 6.26 4.12 4.05 3.41 6.01

d (8.1) brd (8.1) d (15.8) dt (15.8, 6.1) d (6.0) 2H s s s B

156.3 100.4 112.3 nd 107.5 145.2 143.8 131.1 124.8 105.5 148.1 147.9 108.6 119.2 32.6 31.8 71.9 56.1 58.6 101.3

C CH CH CH C C C C CH C C CH CH CH2 CH2

δH 6.79 s 6.96 s 6.63 s

7.32 s

6.87 7.40 2.75 1.94 3.41 4.03 3.36 6.01

d (8.1) d (8.0) t (7.7) dq (7.1, 6.5) t (6.4) s s s



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Figure 1. Morphological changes and inhibition of melanoma cell migration induced by 3 and 4. (A) A2058 melanoma cells were treated with 3 and 4 (5 μM). After 2 h, the cells were fixed and stained with Alexa-488-labeled phalloidin to visualize the cytoskeleton of the cells. Arrows indicate actin stress fibers. Scale bars: 25 μm, 100×: magnification power of the objective. (B) The migration of A2058 melanoma cells was evaluated with the wound-healing assay in the presence or absence of 3 or 4 (2, 5, or 10 μM). The migration of cells was monitored for 24 h by using a Nikon Eclipse Ti-E inverted microscope equipped with a 10× Nikon Plan Fluor objective. Results are shown as percentages of the control (cells migrated during the 24-h experiment) and given as means ± SD (n = 4; *p < 0.01, as assessed by ANOVA and Bonferroni’s post hoc test).

3″) demonstrated the connection of the methoxy group to the C3 unit. The location of this 3″-methoxypropenyl side chain at C-5 was proved by the long-range correlations between C-1″ and H-4 and H-6. The methylenedioxy group on ring B was evidenced by the three-bond HMBC correlations between the proton signal at δH 6.01 and C-3′ and C-4′. The substitution pattern was corroborated by the NOESY cross-peaks between H-3/H-2′, OCH3-7/H-6, H-6/H-1″, H-1″/H-4, H-4/H-3, and OCH3-3″/H-3″. The E geometry of the C-1″/C-2″ olefin was deduced from the coupling constant of 15.8 Hz. All of the above evidence was used to confirm the structure of this compound as 1″-dehydroegonol 3″-methyl ether (1). Compound 2 was obtained as an amorphous solid. Its HRESIMS displayed a quasimolecular ion peak at m/z 363.1202 [M + Na]+, indicating the molecular formula, C20H20O5. Similarly, as in the case of compound 1, the 1H NMR and JMOD spectra showed the presence of two methoxy (δH 4.03 s, 3.36 s, δC 56.1, 58.6) and one methylenedioxy (δH 6.01 s, δC 101.3) substituents in 2. After the 1H and 13C NMR data on 2 had been assigned by analysis of its 1H−1H COSY, HSQC, and HMBC spectra, it became clear that compounds 1 and 2 are based on the same parent system and differ only in the C3 side-chain. The absence of signals of a disubstituted olefin group (C-1″, C-2″) and the appearance of signals of a saturated C3 side-chain (δH 2.75 t, 1.94 dq, 3.41 t, δC 32.6, 31.8, 71.9) indicated the presence of a 3″-methoxypropyl group in 2, in contrast with the 3″-methoxypropenyl residue in 1. All of the above data were compatible with the egonol 3″-methyl ether structure of compound 2.

Compound 1 was isolated as an amorphous solid. It was shown by HRESIMS to have a molecular formula of C20H18O5 according to the quasimolecular ion peak at m/z 361.1045 [M + Na]+ (calcd for 361.1052 C20H18O5Na). The 1H and JMOD spectra of 1 revealed the presence of two methoxy groups (δH 4.05 s, 3.41 s, δC 56.1, 56.9) and one methylenedioxy group (δH 6.01 s, δC 101.3) (Table 1). Additionally, the NMR spectra exhibited signals attributed to a 17 carbon-containing skeleton involving eight quaternary carbons, eight methines, and one methylene groups. A thorough evaluation of its 1D- and 2DNMR spectra revealed that this compound has a 2arylbenzofuran-type norneolignan structure. One trisubstituted aromatic ring (ring B) was demonstrated by the signals of an ABX spin system [δH 7.32 d (J = 0.7 Hz) (H-2′), 6.87 d (J = 8.1 Hz) (H-5′), and 7.40 brd (J = 8.1 Hz) (H-6′)]. The other aromatic ring (ring A) was concluded to be tetrasubstituted according to the aromatic protons at δH 7.14 brs (H-4) and 6.87 brs (H-6). The 1H−1H COSY spectrum showed the presence of an unsaturated C3 unit with correlated protons at δH 6.67 d (J = 15.8 Hz) (H-1″), 6.26 dt (J = 15.8 and 6.1 Hz) (H-2″), and 4.12 d (6.0 Hz) (H-3″). The quaternary carbon at δC 156.3 was assigned as C-2, and the isolated methine at δH 6.82 s and δC 100.5 as C-3 taking into consideration the longrange C−H correlations observed in the HMBC spectrum between C-2 and H-3 and H-2′, and between the H-3 and C-9 and C-8. The correlation of the methoxy signal at δH 4.05 with the carbon signal δC 145.2 (C-7) indicated the presence of the methoxy group at C-7. Similarly, the HMBC cross-peak of the methoxy signal at δH 3.41 with the carbon signals at δC 73.2 (CC



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Figure 2. Adhesion of melanoma cells to brain endothelium in the presence of the isolated lignans. A2058 and D3 cells were pretreated with each of the isolated lignans for 3 h, and the melanoma cells were then plated onto a confluent monolayer of the endothelial cells in the presence of the same compound and left for 80 min. (A) Each of the isolated compounds were used individually in a concentration of 10 μM. For evaluation of the effect of 3, melanoma cells were pretreated with 5 μM, D3 cells were pretreated with 2 μM, and during the attachment of melanoma cells 2 μM 3 was used. B. Five different experimental groups were used: D3+A2058: untreated control; D3(3)+A2058: adhesion of untreated A2058 cells on 3 (2 μM)pretreated D3 cells; D3+A2058(3): adhesion of 3 (5 μM)-pretreated A2058 cells; D3(3)+A2058+(3): adhesion of untreated A2058 cells on 3 (2 μM)-pretreated D3 cells in the presence of 3 (2 μM); D3(3)+A2058(3)+(3): adhesion of 3 (5 μM)-pretreated A2058 cells on pretreated D3 cells (2 μM) in the presence of 3 (2 μM). Results are presented as percentages of the control and given as means ± SD (n = 3; *p < 0.05, **p < 0.01 as assessed by ANOVA and Bonferroni’s post hoc test).

Cell Viability Assay. In the first set of experiments, the isolated lignans (1−6) were tested on the viability of cerebral endothelial cells and melanoma cells. A2058 human metastatic melanoma cells, hCMEC/D3 human brain endothelial cells, and primary rat brain endothelial cells (RBECs) were exposed to various concentrations (1−10 μM) of these lignans for 8 h. There were no significant changes in the viability of the brain endothelial and melanoma cells (Figure S13, Supporting Information), indicating that the experimental results were not influenced by possible cytotoxic effects of the tested compounds. Morphological Changes and Inhibition of Migration of Melanoma Cells. Phase contrast microscope images (Figure S14A, Supporting Information) revealed that 3 (5 μM) resulted in numerous melanoma cells which had an elongated (spindle-like) shape and longer protrusions, and treatment with 4 changed the morphology of the melanoma cells. To visualize the morphological and cytoskeletal changes, cells treated with 3 or 4 (each 5 μM) were stained with Alexa488-conjugated phalloidin [Figure 1A (100×), Figure S14B (40×), Supporting Information]. Exposure to 3 resulted in many cells with elongated morphology with actin-rich protrusions, filopodia, and actin stress fibers. In contrast, the

Compound 3 was identified as helioxanthin by comparison of its spectroscopic data with those published in the literature.29 Helioxanthin has been isolated from several genera, and it is the most widely investigated lignan from the Heliopsis genus.30−32 Compound 4 was identified on the basis of UV, ESIMS, and 1D- and 2D-NMR spectroscopy as (7E)-7,8-dehydroheliobuphthalmin. This compound has earlier been reported from Heliopsis bupthalmoides, although without 13C NMR spectroscopic data.8 In 2D-NMR experiments, the chemical shifts assignment of all the carbons were assigned, as listed in the Experimental Section. It has also been described from Biota orientalis leaves.33 By means of APCIMS and 1H and 13C NMR spectroscopy and comparison of the measured data with published values,34 compound 5 was determined to be heliobuphthalmin with the threo configuration at C-8 and C-8′. This compound has also been identified from the whole plant of Justicia ciliata35 and the stem bark of Pycnanthus angolensis.34 From a consideration of its APCIMS data and 13C NMR chemical shift values36 compound 6 was found to be identical with 7-acetoxyhinokinin. The complete assignments of the 1H NMR data were determined by 2D-NMR analysis, as listed in the Experimental Section. D



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Figure 3. Effects of 3 and 4 on the barrier function of the BBB. Measurement of TEER (A). Data are presented as the percentage resistance of untreated RBECs measured 2 h after treatment. Results are presented as percentages of the control and given as means ± SD (n = 3; *p < 0.05, **p < 0.01 as assessed by ANOVA and Bonferroni’s post hoc test). ZO-1 immunofluorescence staining of RBECs (B) and D3 cells (C). After the 2-h induction of the endothelial layer with the compounds, the cells were stained with anti-ZO-1 antibody (red), a marker of endothelial cells tight junction (TJ) and with Hoechst 33342 (blue) as nuclear staining. Scale bars: 100 μm, 10×, 20×: magnification power of the objective.

mice.16,27 Although 3 and 4 induced different morphological changes, both compounds inhibited the migration of the melanoma cells. Others have shown that enterolactone treatment resulted in morphological changes with fewer filopodia and inhibited the migration of breast cancer cells.22 Furthermore, schisandrin B and honokiol reversed the TGF-βinduced elongated morphology, eliminated the filopodia, and enhanced the migration of the cancer cells.17,37 The effects of these lignans on the filopodia and migration may be consistent with the changes by 4, since this compound gave rise to an expanded morphology different from the mostly rounded shape of the cells induced by the above-mentioned lignans. However,

presence of 4 induced melanoma cells with a more expanded shape without large filopodia. A wound-healing assay was used to investigate the effects of 3 and 4 on the directional migration of melanoma cells. Both lignans reduced the rate of migration of A2058 cells (Figure 1B). The presence of 10 μM 4 reduced the migration of the melanoma cells most effectively. Other lignans have also been shown to inhibit the migratory properties of different cancer cells. Schisandrin B17 and enterolactone22 suppressed breast cancer cell migration, honokiol reduced nonsmall cell lung cancer migration,14 and picropodophyllin inhibited the migration of uveal melanoma cells in vitro.16 Some of these lignans effectively inhibited the metastasis of cancer cells in E



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Figure 4. Endothelial cell migration induced by 3 and 4. The migration of D3 endothelial cells was evaluated with the wound-healing assay for 24 h. Results are shown as percentages of the control (cells migrated during the 24-h experiment) and given as means ± SD (n = 4; *p < 0.05, **p < 0.01 as assessed by ANOVA and Bonferroni’s post hoc test).

expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin of human umbilical vein endothelial cells (HUVECs) activated with TNF-alpha or other reagents is downregulated by lignans, including honokiol,25 sauchinone,24 or sesaminol-6-catechol.40 Similar effects were observed in human aortic endothelial cells treated with different lignans.18,23,26 These investigations are in line with the present findings suggesting that certain lignans influence the adhesion properties of different types of endothelial cells. Further investigations are needed to identify the adhesion molecules affected by 3 on cerebral endothelial cells. The presented results suggest the key role of endothelial cells in the reduction of melanoma attachment caused by treatment with 3. Improvement of the Barrier Function of Cerebral Endothelial Cells. The cerebral endothelial cells forming the BBB have a crucial role in hindering the entry of solutes and cells into the brain. Hence, further characterization of the effects of the tested lignans on the barrier function of the endothelial layer was undertaken. The BBB integrity was evaluated via measurement of the transendothelial electrical resistance (TEER), which is a widely used marker of the tightness of the paracellular junctions. Then, 2 h after the treatment of a monolayer of primary RBECs with 5 μM of 3, the TEER was significantly elevated by about 15−20% relative to the control. A minor elevation of the TEER was likewise observed in the presence of 4 (5 μM) as well (Figure 3A). Continuous lines of tight junctions between brain endothelial cells are essential for the integrity of the BBB. Therefore, the expression and localization of ZO-1 were investigated, which is an important protein component of tight junctions (Figure 3B,C, and Supporting Information, Figure S15). Immunofluorescence studies revealed a stronger ZO-1 staining at the cell−cell contacts between adjacent endothelial cells in the presence of 5 μM of 3, and a similar effect was seen when the cells were treated with 4. Furthermore, even 2 μM of 3 resulted in a more pronounced and continuous staining of ZO-1 on the edge of the human endothelial D3 cells than in the control D3 culture. This suggests that 3 can stimulate human cerebral endothelial cells in lower concentrations than in the case of RBECs. Primary RBECs are able to better preserve the barrier characteristics than are cell lines, and besides possible species differences, this might cause slight differences in the observed effects of the lignans on the tight junctions. Moreover, 4, which

the mechanisms of inhibition of melanoma cell migration induced by 3 and 4 have not been clarified so far. Interaction of Melanoma and Brain Endothelial Cells. Different lignans have been reported to reduce the expression of cell adhesion molecules,18,24−26 while their direct effects on cell adhesion (leukocytes or tumor cells) to the endothelium remain largely unexplored. Adhesion to the endothelial cells forming the blood vessels of the brain vasculature is the first crucial step of the extravasation and the formation of brain metastasis of melanoma cells.38 Therefore, the ability of the isolated compounds to inhibit the attachment of melanoma cells to brain endothelial cells was investigated. For this, D3 human brain endothelial cells and A2058 human melanoma cells were pretreated with the different lignans for 3 h and then treated with the same lignan during the adhesion assay. Compound 3 (2 μM) decreased the number of melanoma cells adhering to the endothelial layer, while the other compounds had no effect (Figure 2A). There were no differences observed in the case of treatment with the other lignans even when the cells were treated with a higher concentration of the compounds (10 μM), and therefore subsequent experiments were performed only with 3 and 4. In order to determine the cell-type-specific impact of 3, either melanoma or endothelial cells were pretreated with the compound, and adhesion experiments were performed in the absence of the lignan (Figure 2B). Incubation of the melanoma cells with 5 μM of 3 did not have any effect on the adhesion. However, when the brain endothelial cells were pretreated with 2 μM of 3, the extent of adhesion of the melanoma cells to the brain endothelium decreased significantly but to a lesser extent than that observed when this lignan was present during the adhesion experiment. In the latter case, pretreatment was not able to reduce the adhesion further. These findings indicate a specific role of the endothelial changes induced by 3 in reducing the adhesion, and these changes are reversible. Although both 3 (5 or 10 μM) and 4 (2−10 μM) induced morphological changes in the melanoma cells, which could additionally influence the adhesive properties,39 in the experimental setup used the investigated lignans did not affect the adhesion properties of this cell type. A large number of studies have shown that integrins, cellular adhesion molecules, and selectins of endothelial cells are among the molecules that may affect tumor adhesiveness to endothelia.38 Previous investigations have shown that the F



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experiments, gradient-enhanced versions were used. Mass spectrometric (MS) measurements were carried out in positive ionization mode with an API 2000 MS/MS instrument (AB SCIEX, Framingham, MA, USA) equipped with an atmospheric pressure chemical ionization (APCI) interface. The source temperature was 400 °C. Data acquisition and evaluation were performed with Analyst 1.5.2 software. For vacuum-liquid chromatography (VLC), SiO2 (silica gel 60 GF254, 15 μm, Merck, Darmstadt, Germany) and reversed-phase SiO2 (LiChroprep RP-18, 40−63 μm, Merck) were applied. Separations were monitored by TLC (aluminum sheets coated with silica gel 60 F254, 0.25 mm, Merck 5554, and silica gel 60 RP-18 F254s, Merck) plates. The chromatograms were visualized at 254 and 366 nm, and by spraying with concentrated H2SO4, followed by heating at 110 °C. Medium-pressure liquid chromatography (MPLC) was performed with a Büchi apparatus (Büchi Labortechnik AG, Flawil, Switzerland), using a 40 × 75 mm RP18ec column (Büchi, 40−63 μm). Preparative thin-layer chromatography was carried out on silica gel 60 F254 (0.25 mm, Merck), and centrifugal planar chromatography (CLC) was performed with a Chromatotron instrument (model 8924, Harrison Research, Palo Alto, CA, USA) on manually coated SiO2 (silica gel 60 GF254, Merck 7730) plates. HPLC analysis was carried out on a Waters 600 system (Waters Corp., Milford, MA, USA), equipped with a UV detector and an online degasser, using normal or reversed-phase C18 columns (LiChroCART 5 μm, 100 Å, 250 × 4 mm column, Merck) at 25 °C. Plant Material. The roots of H. helianthoides var. scabra “Asahi” were obtained from a nursery (Hegede Flower Nursery Ltd., Kecskemét, Hungary, identified by Dóra Rédei, University of Szeged, Department of Pharmacognosy) in the flowering period in September 2009. A voucher specimen (No. 819) has been preserved in the Herbarium of the Department of Pharmacognosy, University of Szeged, Szeged, Hungary. The plant material was washed, cleaned, and processed in a fresh form. Extraction and Isolation. The fresh roots of H. helianthoides var. scabra were extracted with MeOH (90 L) at room temperature. After evaporation, the MeOH extract (after dilution with H2O) was subjected to solvent partitioning to obtain CHCl3- and H2O-soluble fractions. The lignan-containing CHCl3 phase was concentrated under a vacuum, yielding 80 g of material, which was subjected in two parts to silica gel VLC (2 × 250 g), using a gradient system of n-hexane− EtOAc (10:0, 9:1, 8:2, 7:3, 1:1, and 0:10). In total, 78 fractions were collected and combined into 12 main fractions (I−XII) with regard to the results of TLC monitoring. Lignan-containing fractions (VIII−IX, 9 g) were selected for further purification, which was carried out by a combination of RP-VLC, using MeOH−H2O (8:2, 85:15, 9:1, 10:0) and then MeOH−EtOAc (9:1, 6:4, 10:0) gradients. From a total of 75 fractions, fractions 35−40 (1.15 g), 41−46 (668 mg), and 17−22 (470 mg) were selected for further separation. Fractions 35−40 were pooled and purified by MPLC with a gradient system of n-hexane−EtOAc (1:0, 9:1, 8:2, 7:3, 6:4, 1:1, 0:1) at a flow rate of 60 mL/min. It was separated into 45 subfractions, among which subfractions 25−29 were subjected to multiple CLC (2 mm silica gel CLC plate, gradient system of benzene−CH2Cl2−Et2O, 9:3:0, 6:3:0, 6:3:0.5; 5 mL/min flow rate). The fractions obtained were finally purified by preparative TLC carried out in a fumehood (benzene−CH2Cl2−Et2O, 6:3:1), which resulted in the purification of compounds 4 (13 mg) and 5 (20 mg). Fractions 41−46 were pooled and purified by MPLC with a gradient system of n-hexane−EtOAc (1:0, 9:1, 8:2, 7:3, 6:4, 1:1, 0:1) at a flow rate of 60 mL/min. This fraction was separated into 78 subfractions, among which 7−19 and 20−36 were selected for further separations. Subfractions 7−19 were pooled and subjected to preparative TLC (n-hexane−EtOAc, 6:4), and RP-HPLC (MeCN− H2O, 8:2) of the almost pure fractions resulted in the isolation of 1 (6 mg) and 2 (2 mg). Subfractions 20−36 were chromatographed on a 1 mm silica gel CLC plate with a gradient system of n-hexane−Me2CO (9:1, 8:2, 1:0) at a flow rate of 3 mL/min. Final purification was conducted by HPLC (n-hexane−EtOAc, 8:2) to give compound 3 (2 mg).

also increased the TEER, enhanced the presence of ZO-1 in the junctions of the cells at both 2 and 5 μM. This appears to be the first evidence of the barrier-enhancing effect of a lignan. Lignans, as one of the major classes of phytoestrogens, are structurally similar to estrogen. Both estrogen and the steroid hormone hydrocortisone enhance the tightness of the BBB by upregulating another tight junction protein, claudin-5.41,42 Previously it was demonstrated that TEER of RBECs decreased in the presence of A2058 and B16/F10 melanoma cells.43 It is therefore tempting to assume that treatment with 3 or 4 may contribute to a reduction in the rate of transmigration of melanoma cells by strengthening the barrier function of the cerebral endothelial cells. Besides contributing to a lowering of the transmigration rate, the barrier-strengthening effects of 3 and 4 may be of clinical relevance in other pathologies. Several pathological conditions of the CNS are associated with the opening of the BBB, and lignans might prove to be protective against this phenomenon by strengthening the tight junctions of cerebral endothelial cells. However, further investigations are needed to test whether compounds 3 and 4 can protect the tight junctions in pathological conditions. Effects on the Migration of Endothelial Cells. An important step in the growth of metastases is the vascularization of the tumor. In this angiogenic process, the migration of endothelial cells is a key element. As the next step, therefore the effects of 3 and 4 on the migration of brain endothelial cells by using the wound healing assay were assessed. As shown in Figure 4, each of these compounds led to a significant reduction in endothelial cell migration. This appeared to be a concentration-dependent effect, with 10 μM of 3 and 10 μM of 4 displaying a more pronounced influence on the migration than 5 and 2 μM, respectively. This trend is reminiscent of the concentration-dependent mitigation of melanoma cell migration evoked by lignans 3 and 4. After diapedesis, close contact to the basolateral side of endothelial cells is essential for the proliferation of metastatic cells.38 Metastatic tumor growth is therefore often associated with the formation of tumor vasculature, i.e., tumor angiogenesis.44 One of the possible strategies to prevent tumor angiogenesis is to inhibit endothelial cell migration into the tumor. Interestingly, secoisolariciresinol diglucoside, a plant lignan isolated from flaxseed, exerts an angiogenic effect mediated with increased levels of VEGF and Ang-1 protein in human coronary arteriolar endothelial cells.45 Moreover, the same effect of this lignan was demonstrated in vitro in HUVECs without the induction of vascular permeability and the upregulation of ICAM-1 and VCAM-1 expression.46 In contrast, another study has indicated the antiangiogenic property of honokiol, with a pronounced downregulation of VEGF and ICAM-1 in H1299 human lung adenocarcinoma cells treated with this lignan.47 Apart from the complexity of the roles of different lignans in angiogenesis, the present in vitro experiments indicate that 3 and 4 may have antiangiogenic properties.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined in CHCl3 with a Perkin-Elmer 341 polarimeter (Waltham, MA, USA). NMR spectra were recorded in CDCl3 on a Bruker Avance DRX 500 spectrometer (Bruker, Fallanden, Switzerland) at 500 MHz (1H) or 125 MHz (13C). 2D data were acquired and processed with standard Bruker software. For 1H−1H COSY, HSQC, and HMBC G



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Pooled fractions 17−22 were also chromatographed on a 1 mm silica gel CLC plate with a gradient system of n-hexane−EtOAc (9:1, 8:2, 7:3, 0:1) at a flow rate of 3 mL/min. Finally, the fractions obtained were preparative TLC (benzene−CH2Cl2−Et2O, 1:1:1), which resulted in compound 6 (4 mg). 1″-Dehydroegonol 3″-Methyl Ether (1). Amorphous solid; for 1H and 13C NMR data, see Table 1; APCIMS (positive mode) m/z 339 [M + H]+, 307 [(M + H) − CH3OH]+, 279 [(M + H) − CH3OH − CO]+, 249, 221, 149; HRESIMS m/z 361.1045 [M + Na]+ (calcd for C20H18O5Na, 361.1052). Egonol 3″-Methyl Ether (2). Amorphous solid; for 1H and 13C NMR data, see Table 1; APCIMS (positive mode) m/z 341 [M + H]+, 309 [(M + H) − CH3OH]+, 281 [(M + H) − CH3OH − CO]+, 251, 223, 149; HRESIMS m/z 363.1202 [M + Na]+ (calcd for C20H20O5Na, 363.1208). Helioxanthin (3). Yellow substance with a strong blue fluorescence at 360 nm; 1H and 13C NMR data identical with those published by Lee et al.;29 APCIMS (positive mode) m/z 349 [M + H]+, 319 [(M + H) − CH2O]+, 305, 301, 291, 275. (7E)-7,8-Dehydroheliobuphthalmin (4). Colorless oil; [α]24D −175 (c 0.1, CHCl3); 1H NMR data identical with published data;813C NMR (CDCl3, 125 MHz) δC 173.1 (C-9′), 167.1 (C-9), 149.5, 147.7 (C-1, C-1′, C-2, C-2′), 142.5 (C-7), 133.2 (C-4′), 129.5 (C-8), 128.8 (C-4), 122.5 (C-5), 122.1 (C-5′), 109.4 (C-3′), 108.4 (C-3), 108.2 (C-6), 107.8 (C-6′), 101.2 (1,2-OCH2O−), 100.7 (1′,2′-OCH2O−), 52.2 (OCH3-9′), 52.0 (OCH3-9), 45.5 (C-8′), 35.7 (C-7′). Heliobuphthalmin (5). Amorphous solid; [α]24D −3 (c 0.2, CHCl3); 1H and 13C NMR data identical with those published by Abrantes et al.;34 APCIMS (positive mode) m/z 415 [M + H]+, 383 [(M + H) − CH3OH]+, 351, 333, 135. 7-Acetoxyhinokinin (6). Amorphous solid; [α]26D −46 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz) δH 6.73 (1H, d, J = 7.9 Hz, H5′), 6.65 (1H, s, H-2), 6.65 (1H, d, J = 7.9 Hz, H-6′), 6.64 (1H, d, J = 7.9 Hz, H-6), 6.36 (1H, d, J = 7.9 Hz, H-5), 6.34 (1H, s, H-2′), 6.14 (1H, d, J = 3.4, H-7), 5.97, 5.96 (each 1H, s, −OCH2O−), 5.93, 5.92 (each 1H, d, J = 1.0 Hz, −OCH2O−), 4.29 (1H, t, J = 8.7 Hz, H-9′a), 3.97 (1H, dd, J = 8.7, 5.6 Hz, H-9′b), 2.81 (1H, m, H-8′), 2.70 (1H, dd, J = 6.1, 3.4 Hz, H-8), 2.46 (1H, dd, J = 13.8, 8.2 Hz, H-7′a), 2.35 (1H, dd, J = 13.8, 7.4 Hz, H-7′b), 2.13 (3H, s, OAc); APCIMS (positive mode) m/z 413 [M + H]+, 353 [(M + H) −CH3COOH]+. Cell Culture and Treatments. The human microvascular cerebral endothelial cell line (hCMEC/D3; abbreviated as D3) was maintained in EBM-2 medium (Lonza, Basel, Switzerland) supplemented with EGM-2 growth factors (Lonza) and 5% fetal bovine serum (FBS, Sigma-Aldrich, Munich, Germany). The A2058 human melanoma cell line (obtained from the European Collection of Cell Cultures) was cultured in MEM (Sigma-Aldrich) and 5% FBS. For TEER measurements, primary RBECs were used because of their superior barrier characteristics. RBECs were isolated from 2-week-old rats, as described previously.48,49 Briefly, after removal of the meninges, the cerebral cortices were cut into small pieces and digested in two steps with collagenase and collagenase/dispase, followed by centrifugation on a Percoll gradient. Isolated microvessels were plated on fibronectin/collagen-coated dishes. Endothelial cells growing out of the microvessels were cultured in DMEM/F12 (Life Technologies, Carlsbad, CA, USA), 10% plasma-derived serum (PDS, FirstLink, Wolverhampton, UK), and growth factors. In the first 2 days, 4 μg/mL puromycin was added to remove contaminating cells. Before being tested biologically, the purity of the isolated lignans was checked by TLC and HPLC. The compounds were dissolved in DMSO as 10 mM stock solutions. Control cells received an equivalent concentration of DMSO as vehicle. The DMSO concentration was 0.1% (v/v) in all cases. Cell Viability Assay. The effects of the lignans on the viability of the melanoma and endothelial cells were quantified with the EZ4U assay (Biomedica, Vienna, Austria), a colorimetric assay of the activity of mitochondrial dehydrogenases, which correlates with the number of living cells.50 A2058, D3, and RBE cells were seeded in 96-well plates. After confluency had been reached, the cells were treated for 8 h with 10, 5, 2.5, or 1.25 μM of each of the individual isolated compounds, in

serum-free MEM (A2058), in serum-free EBM-2 (D3), or in DMEM/ F12 and 5% PDS (RBEC). After incubation with EZ4U for 45 min, the absorbance (OD at 450 nm) was detected by FLUOstar Optima (BMG LABTECH, Ortenberg, Germany). Phalloidin Staining of the Actin Cytoskeleton. Following treatment for 2 h with 3 or 4 (5 μM), A2058 melanoma cells were fixed with 4% HCHO and permeabilized with Me2CO at −20 °C for 10 min. After blocking, cells were stained with Alexa488-phalloidin (Life Technologies, Budapest, Hungary). Mounting was performed in antifading embedding medium (Biomeda, Burlingame, CA, USA), and the distribution of the signal was studied with a Nikon Eclipse TE2000U photomicroscope (Nikon, Vienna, Austria) with epifluorescent capabilities connected to a digital camera (Spot RT KE). Attachment of Melanoma Cells to Brain Endothelial Cells. D3 human brain endothelial cells were grown until confluency in 12well or 24-well plates. Endothelial cells and melanoma cells were pretreated with the studied lignans for 3 h. After pretreatment, A2058 melanoma cells were fluorescently labeled with Oregon Green 488 carboxylic acid diacetate succinimidyl ester (Life Technologies), using the protocol supplied by the manufacturer. In total, 105 (12-well plate) or 5 × 104 (24-well plate) melanoma cells/well were loaded onto the endothelial monolayer in serum-free medium and left for 80 min. After washing, the cells were fixed with EtOH−AcOH (95:5) at −20 °C for 5 min. Melanoma cells attached to endothelial cells were photographed and counted with the Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). Measurement of TEER. Brain endothelial tight junctions form a continuous line and restrict the free paracellular passage of even ions. Therefore, an electrical resistance (transendothelial electrical resistance, TEER) is formed between the apical and basolateral sides of the monolayer, which is a widely accepted indicator of the barrier properties of brain endothelial monolayers. RBECs were grown on collagen/fibronectin-coated semipermeable filters (0.4 mm pore size, 1.12 cm2, Costar Corning Transwell, Tewksbury, MA, USA). After reaching confluence, the endothelial monolayer was supplied with 550 nM hydrocortisone, 250 μM CPT-cAMP (Sigma-Aldrich), and 17.5 μM phosphodiesterase inhibitor RO-201724 (Hoffmann-La Roche, Basel, Switzerland) and placed into the wells of the CellZscope instrument (nanoAnalytics, Münster, Germany) containing astrocyteconditioned medium. TEER measurements were performed automatically. After TEER had reached its plateau (120−140 Ω·cm2), the endothelial cells were treated with 3 (2.5 or 5 μM) or 4 (5 μM), and TEER was followed for 2 h. Immunofluorescence Staining. D3 cells and RBECs were cultured until confluency in 96-well plates. The cells were treated for 2 h and then fixed with EtOH−AcOH (95:5) at −20 °C for 5 min. After blocking with 3% BSA in PBS for 30 min, samples were incubated with primary rabbit antibody against ZO-1 (Invitrogen, Carlsbad, CA, USA) overnight. The staining was visualized with Cy3 conjugated antibody (goat anti-rabbit, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). After immunostaining, nuclear staining of the cells was carried out with Hoechst 33342 (SigmaAldrich). Images were recorded by a Nikon Eclipse TE2000U photomicroscope with epifluorescent capabilities connected to a digital camera (Spot RT KE). Wound-Healing Assay. D3 endothelial cells were seeded into 12well tissue culture plates coated with rat tail collagen. The cells were grown to 100% confluency, and the monolayer was then wounded by scratching with a pipet tip, washed with PBS, and exposed to treatment in Leibovitz medium supplemented with 5% FBS. Cells were monitored over 24 h, and phase contrast images were taken with an Andor NEO sCMOS camera (Andor Technology, Belfast, UK) connected to the Nikon Eclipse Ti-E inverted microscope (Nikon Instruments) equipped with a home-built incubator set to 37 °C and a 10× Nikon Plan Fluor objective, all placed onto a Prior Proscan II motorized stage (Prior Scientific Instruments, Cambridge, UK). The wound-healing effect was quantified by averaging the number of migrating cells counted in five wounded areas. H



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S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +36-62546453. Fax: +36-62-545704. Author Contributions

⊥ Z.H. and J.H. contributed equally to this work, and both are named as first authors.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of the TÁ MOP 4.2.4.A/2-11-1-2012-0001 “National Excellence Program”. This work was supported by the New Hungary Development Plan Projects TÁ MOP4.2.2.A-11/1/KONV-2012-0035, TÁ M OP-4.1.1.C-12/1/ KONV-2012-0014, TÁ MOP-4.2.2.A-11/1/KONV-2012-0052, Hungarian Scientific Research Fund (OTKA PD-100958, K100807, and K109846), and the National Development Agency (Hungary-Romania Cross-Border Co-operation Programme 2007−2013: HURO/1101/173/2.2.1). I.W. was supported by a János Bolyai Research Fellowship. We thank Péter Galajda for the use of the Nikon Eclipse Ti-E inverted microscope (Nikon).

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Evaluation of Lignans from Heliopsis helianthoides var. scabra for

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