Diselaginellin B, an Unusual Dimeric Molecule from Selaginella

Article pubs.acs.org/jnp

Diselaginellin B, an Unusual Dimeric Molecule from Selaginella pulvinata, Inhibited Metastasis and Induced Apoptosis of SMMC7721 Human Hepatocellular Carcinoma Cells Yuan Cao,†,‡,§ Ming Zhao,‡ Yue Zhu,‡ Zhen-Hua Zhu,‡ Lukas Oberer,⊥ and Jin-Ao Duan*,‡ †

Department of Pharmacy, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210036, People’s Republic of China ‡ Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China § Department of Pharmacology, University of California, Irvine, Irvine, California 92697, United States ⊥ Novartis Institute of Biomedical Research (NIBR), 4056 Basel, Switzerland S Supporting Information *

ABSTRACT: Two new unusual dimeric selaginellins, diselaginellins A and B (1 and 2), along with two known derivatives, selaginellin (3) and selaginellin B (4), were isolated from Selaginella pulvinata. Their structures were elucidated by extensive NMR and high-resolution ESIMS data analysis. Compound 2 displayed apoptosis-inducing and antimetastatic activities against the human hepatocellular carcinoma cell line SMMC-7721. A microarray analysis revealed that genes related to metabolism, angiogenesis, and metastasis were altered by 2. The up- and down-regulation of the mRNA levels of related genes was confirmed by RT-qPCR. Metabolism modulation and metastasis inhibition might be the mechanisms of the antitumor properties of diselaginellin B (2).

H

fraction was found to be cytotoxic against the human hepatocellular carcinoma cell line (SMMC-7721). This investigation led to the isolation of two unprecedented etherlinked dimers, diselaginellins A (1) and B (2), along with two known derivatives, selaginellin (3) and selaginellin B (4). Herein, the isolation and structural elucidation of the new compounds are reported. The apoptosis-inducing and metastasis-inhibitory activities, as well as the targeted genes of SMMC-7721 cells affected by diselaginellin B (2), are discussed.

uman hepatocellular carcinoma (HCC) is among the most common and lethal malignancies worldwide.1 Its endemic prevalence in Asia, especially in China, accounts for the high mortality of HCC in this region. Given its high probability of recurrence and metastasis,2 HCC prognosis remains extremely poor. Moreover, current chemotherapy protocols for HCC are known to induce chemoresistance and have a range of adverse effects. Therefore, the development of novel drugs for HCC treatment is urgently required. Plantderived natural products continue to be a valuable resource for the discovery of novel cancer chemopreventive agents. Selaginella pulvinata (Hook. et Grev.) Maxim., a perennial herb that primarily grows in southwest China, is listed as one source of the officinal Selaginellae Herba in the Chinese Pharmacopoeia (2015 edition).3 This herb has been extensively used in oriental medicine as an anticancer, anti-inflammatory, and antidiabetic agent.4 The earliest use of S. pulvinata for the treatment of tumors dates back to 2737 B.C., which is well documented in “Shen Nong Ben Cao Jing” (The Divine Farmer’s Materia Medica). Previous studies of this genus have resulted in the discovery of 30 selaginellins, a rare group of phenolic compounds. Many of these compounds are receiving increased attention due to their biological activities, such as cytotoxicity, inhibition of phosphodiesterase-4, and neuroprotection.5,6 In our continuing research to explore the bioactive components from this plant, the n-BuOH-soluble © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Diselaginellin A (1) was obtained as a red powder. A molecular formula of C68H46O9 with 46 indices of hydrogen deficiencies was deduced from high-resolution (HR)-ESIMS (m/ z1007.32195 [M + H]+; calcd for C68H47O9+, 1007.32146) and 13C NMR data. UV absorptions at 270, 300, and 420 nm were indicative of a selaginellin chromophore.5 The IR spectrum showed absorption bands for hydroxy (3414 cm−1), alkynyl (2200 cm−1), and aromatic (1596, 1513 cm−1) functionalities, respectively. The 1H NMR spectroscopic data (Table 1) indicated the presence of two aromatic AX-spin systems (δH 7.65 and 7.36, d, Received: May 9, 2017 Published: November 16, 2017 3151

DOI: 10.1021/acs.jnatprod.7b00404 J. Nat. Prod. 2017, 80, 3151−3158

Journal of Natural Products

Article

Chart 1. Structures of Compounds 1−4

J = 8.1 Hz; δH 7.71 and 7.36, d, J = 8.1 Hz), originating from the ortho-tetrasubstituted A- and A′-rings, respectively; and five AA′XX′ systems (J = 8.8 Hz for all systems), representing the protons of the para-substituted B-, B′-, D′-, E-, and E′-rings. These spin systems were revealed by DQF-COSY analysis. The 13 C NMR data (Table 1) showed signals at δC 83.8 (2C), 98.8, and 99.0, indicative of two acetylenic moieties. These moieties were connected to rings A/B and A′/B′, respectively, as evidenced by the HMBC correlations between H-28/32 and C27, as well as H-28′/32′ and C-27′, which was supported by ROESY correlations of H-28 with H-34, as well as H-28′ with H-34′ (Figure 2). The protons of either the C- or D-ring merged into two broad signals at δH 6.49 (4H, overlap, H-2,6 and H-9,11) and 7.05 (4H, overlap, H-3,5 and H-8,12), respectively, which could be interpreted in terms of a quinone methide (QM)−phenol tautomeric equilibrium at room temperature.5c,i Likewise, tautomerism caused the signals for C-1 and C-10 to be obscured by the baseline. On the other hand, the protons of an additional cyclohexadienone moiety resonating as an ABXY system (δH 6.46, 7.47, 7.25, 6.41, each 1H, dd, J = 10.1, 2.0 Hz) in the 1H NMR spectrum were ascribed to the C′-ring. The well-resolved signals for the C′and D′-rings suggested that C-10′ was involved in the formation of an interselaginellin bond, since etherification of HO-10′ could quench the tautomerism and produce welldispersed signals.7 The linkage between C-34 and C-10′ via an oxygen atom was evidenced by the HMBC correlations from H2-34 to C-10′, C-14, C-15, and C-16, as well as the ROESY correlations of H2-34 with H-16, H-9′, and H-11′ (Figure 1). The conspicuously deshielded H2-34 resonances (δH 5.28 and 5.34, respectively) compared with the corresponding shifts of the chemically equivalent protons in monomers, such as selaginellin5a and selaginellins D,5c M,6c and P,5i were consistent with their chemical environments. Thus, the 2D structure of this unprecedented 34,10′-coupled diselaginellin A (1) was determined as shown in Figure 1. Diselaginellin B (2) was obtained as a red powder. Its molecular formula was defined as C68H46O9 by the HR-ESIMS ion at m/z 1007.32122 [M + H]+ (calcd 1007.32146) and 13C

NMR data. The UV and IR spectra were similar to those of 1, exhibiting the characteristic absorption pattern of selaginellins. The 1H and 13C NMR spectroscopic data of compound 2 were also similar to those of 1, indicating its dimeric nature. However, signals for the C-, C′-, D-, and D′-rings of 2 were all obscured in the baseline, suggesting QM−phenol tautomerism in both moieties. It was assumed that the tautomerism might be due to the acidic conditions of the isolation and purification processes. Thus, K2CO3 was carefully added to the sample solution until the color changed to purple (pH = 7.5) from red,5a followed by NMR data acquisition. As expected, all formerly broadened signals were sharpened and well-resolved in the spectra measured in DMSO-d6, and the aromatic protons/carbons attached to the QM (C and C′) and phenolic rings (D and D′) became averaged (Table 1). The key HMBC (between H2-34 and C-14, C-15, C-16, and C-30′) and ROESY correlations (between H2-34 and H-16, H-29′, and H-31′) (Figure 1), as well as the deshielding of H2-34 (Δδ 0.40), defined a unique C34−O−C30′ ether linkage. Accordingly, the 2D structure of compound 2 was determined (Figure 1). The chemical shifts of C-1/10 and C-1′/10′ were observed at δC 158.9 and 180.6/181.1 under the moderate acidic and basic conditions, respectively. The studies of Sarma et al.7 and Zhang et al.5a showed a similar phenomenon. The acid−base properties of selaginellins explained the formation and stability of the interconvertible species, as illustrated in Scheme 1. Selaginellin derivatives with QM structural moieties isolated from nature were found to exist as a pair of enantiomers, as shown in the crystal structures, due to either QM−phenol tautomerism or atropisomerism.5a−c In a previous investigation, we used chiral phase separation and experimental and calculated electronic circular dichroism (ECD) data to determine the absolute configuration of nontautomeric monomeric selaginellins, i.e., the R and S configurations for (+)- and (−)-selaginellins, respectively.5i For compounds 1 and 2, since both contain two independent symmetry axes (C-7−C19 and C-7′−C-19′ axes), four chiral diastereoisomers (two diastereomeric racemic pairs) inherently exist for each, which was supported by their optical inactivity and the absence of 3152



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Table 1. 1H and 13C NMR Data of Compounds 1 and 2 in DMSO-d6 1 position 1 2/6 3/5 4 7 8/12 9/11 10 13 14 15 16 17 18 19 20/24 21/23 22 25 26 27 28/32 29/31 30 33 34 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′/12′ 9′/11′ 10′ 13′ 14′ 15′ 16′ 17′ 18′ 19′ 20′/24′ 21′/23′ 22′ 25′ 26′ 27′ 28′/32′ 29′/31′ 30′ 33′ 34′

a

δH

2 δC

δH

a

6.49 (m) 7.05 (m)

a a

6.20 (d, 8.1) 7.00 (d, 8.1)

a a

7.05 (m) 6.49 (m)

a a

7.00 (d, 8.1) 6.20 (d, 8.1)

a a

7.65 (d, 8.1) 7.36 (d, 8.1)

6.82 (d, 8.8) 6.57 (d, 8.8)

6.82 (d, 8.8) 6.60 (d, 8.8)

a: 5.30 (d, 12.5) b: 5.36 (d, 12.5) 6.46 (dd, 10.1, 2.0) 7.47 (dd, 10.1, 2.0) 7.25 (dd, 10.1, 2.0) 6.41 (dd, 10.1, 2.0) 6.88 (d, 8.8) 7.02 (d, 8.8)

7.71 (d, 8.1) 7.36 (d, 8.1)

6.79 (d, 8.8) 6.57 (d, 8.8)

6.99 (d, 8.8) 6.67 (d, 8.8)

4.81 (s) (2H)

Cotton effects in the ECD spectra. In the case of compound 1, the racemization process is concurrently induced by both tautomerism and atropisomerism, since the etherification of HO-10′ can block the tautomerization of the C′- and D′-rings. The subsequent chiral phase HPLC analysis of compound 1 afforded two UV peaks, corresponding to two mirror-image ECD curves (Figure 2). Each of the single peaks (1:1 ratio) comprised of a pair of epimers due to the reversible nature originating from the QM−phenol tautomerism (Scheme 2). The racemization process of compound 2 was derived from the tautomeric equilibrium between the C-/C′- and D-/D′-rings and was a priori a racemic mixture and could not be separated into enatiomers.5i Biosynthesis Pathway. Diselaginellins A (1) and B (2) are the first examples of naturally occurring selaginellin dimers. Both compounds should be the dehydrated products of two molecules of rac-3, which were previously isolated from S. pulvinata,5c and were suggested as biosynthetic monomeric precursors. A putative biogenetic pathway for (R,R)-1 and (R,R)-2 from (R)-3 is proposed in Scheme 3. For compound 1, the dehydration involves HO-34 and HO-10′, while for 2, the dehydration occurs between HO-34 and HO-34′. The proposed biosynthesis process rationalizes the structures and the configurations of the dimers. Bioactivity Assays. The effects of compounds 1−4 on the cell viability of SMMC-7721 were evaluated by an MTT assay. Compound 2 exhibited moderate cytotoxic activity against SMMC-7721 cells, with an IC50 value of 9.0 μM, and induced apoptosis in a concentration-dependent manner (Figure 3A,B). Flow cytometric analysis showed that diselaginellin B (2) arrested the cells at the G1 phase (Figure 3C). All isolates were further studied for their antimetastasis activities on SMMC7721 cells. Cells were incubated with nontoxic doses of compounds for 48 h, and the invasive and migratory behaviors were analyzed using Transwell chamber assays with or without Matrigel. Among the tested compounds, 2 at 0.5, 1, or 2 μM resulted in approximately 0.53-, 0.42-, and 0.39-fold reductions in cell invasion, respectively (Figure 3D,E). As shown in Figure 3D,E, the Transwell assay revealed a similar trend, with 2 limiting the migration of cells through the membrane. These results indicate the potential role of 2 in the suppression of cancer cell migration and invasion and in the induction of apoptosis. Microarray Analysis. To identify the mechanism of the antiproliferative, apoptosis-inducing, and antimetastatic activities of diselaginellin B (2), the alteration of the gene expression profile in SMMC-7721 cells following treatment with 2 via cDNA microarray analysis was investigated. The results demonstrated that 30 and 132 mRNAs were enhanced and reduced, respectively. Pathway analysis using DAVID (http://david.abcc.ncifcrf.gov/summary.jsp) and KEGG (http://www.genome.jp/kegg/pathway.html) suggested that genes related to metabolism, angiogenesis, and metastasis were considerably altered. Therefore, RT-qPCR was performed to evaluate the different gene expression profiles; the relative mRNA levels of cyclooxygenases 1 and 2 (COX-1 and COX-2), nicotinamide N-methyl-transferase (NNMT), tetraspanin-8 (TSPAN8), carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7), and microfibrillar-associated glycoprotein 2 (MAGP2) were clearly down-regulated, whereas the expression of dihydrolipoamide dehydrogenase (DLD) was upregulated (Figure 4B).

123.5 136.1 128.9 129.5 140.7 140.7 129.2 114.8 156.7 130.3 83.8 99.0 132.8 115.6 159.1 111.7 68.2 185.8 129.5 136.1 130.5 140.4 129.2 158.6 132.7 114.4 160.2 130.5 120.9 142.1 128.9 129.5 140.4 140.7 129.5 114.8 156.7 129.6 83.8 98.8 132.8 115.7 159.1 111.7 61.3

7.65 (d, 8.1) 7.34 (d, 8.1)

6.91 (d, 8.0) 6.53 (d, 8.0)

6.53 (d, 8.0) 6.40 (d, 8.0)

5.24 (s) (2H)

6.20 (d, 8.1) 7.05 (d, 8.1) 7.05 (d, 8.1) 6.20 (d, 8.1) 7.05 (d, 8.1) 6.20 (d, 8.1)

7.68 (d, 8.1) 7.40 (d, 8.1)

6. 94 (d, 8.0) 6. 53 (d, 8.0)

7.13 (d, 8.0) 7.00 (d, 8.0)

4.78 (s) (2H)

δC 181.1 123.0 138.4 123.5 163.6 138.4 123.0 181.1 123.5 125.6 136.1 130.4 129.5 142.9 141.4 129.7 115.1 156.7 131.1 84.3 99.7 133.3 116.4 160.3 110.9 69.2 180.6 123.0 138.4 124.0 138.4 123.0 164.1 138.4 123.0 180.6 124.0 121.7 142.8 127.3 130.0 140.8 141.3 129.8 115.1 156.8 131.2 85.0 98.6 133.2 115.6 159.4 115.0 61.6

Unassignable due to broad or obscured signals. 3153



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Figure 1. 1H−1H COSY (bold ), key HMBC (→), and ROESY (↔) of 1 and 2.

the oncogenic function of AEG-1, thereby suggesting a potential target for the treatment of metastatic HCC.14 Increased MAGP2 expression is correlated with angiogenesis and chemoresistance.15,16 Deregulation of CEACAM-7 expression has been implicated in colorectal and gastric oncogenesis.17 Collectively, diselaginellin B (2) may exert its pro-apoptotic and antiproliferative effects on tumor cells by modulating the dysregulated cancer-related metabolic enzymes, thus exerting control over the metabolism of tumors. The data indicate that diselaginellin B (2) has the potential to inhibit angiogenesis and reduce the invasion and metastasis of tumors. Selaginellins are an important group of bioactive compounds of the Selaginella species, whose natural occurrence is hitherto confined to this genus. Owing to its potent medicinal potential, efforts continue to expand upon its chemistry and biology.5,6 Diselaginellins A (1) and B (2) represent the first C−O−C linked selaginellins from the genus Selaginella. Each exists as four diastereoisomers (two diastereomeric racemic pairs). Compound 2 displayed metastasis-inhibitory and apoptosisinducing activities against SMMC-7721 cells. The microarray experiments revealed metabolism modulation and metastasis inhibition effects, suggesting the potential of 2 as a promising lead compound.

Figure 2. HPLC-ECD and HPLC-UV analysis of diselaginellin A (1).

Scheme 1. Different Forms of Compound 2 under Different pH’s

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined using a PerkinElmer polarimeter 341, calibrated under good manufacturing practice conditions. UV spectra were recorded on a Shimadzu 2401-PC spectrophotometer. IR spectra were acquired using a Bruker Tensor 27 FT-IR spectrometer. ECD spectra were recorded using a Jasco J-810 spectrometer with a 1 mm path length cell. NMR spectra were recorded on Bruker AV-600 spectrometers. MS data were acquired on a VG Auto Spec-3000 mass spectrometer and an LTQ Orbitrap XL mass spectrometer (Thermo Scientific). Column chromatography was performed over silica gel H (100−200 mesh, 200−300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China) and Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Semipreparative HPLC was performed on an Agilent 1260 liquid chromatography system equipped with a YMC ODS A column (150 × 10 mm). The HPLC-ECD coupling system was composed of JASCO LC-Net II/ADC chromatography data solutions, a PU-2089 Plus quaternary gradient pump, a CO-2060 Plus intelligent column thermostat, and a CD-2095 Plus chiral detector. Plant Material. The S. pulvinata plants were collected in Dali County, Yunnan Province, China, in October 2012. The identification of the plant was verified by Dr. Prof. Jin-Ao Duan, Nanjing University

COX-1/COX-2-controlled prostaglandin metabolism has been implicated in the pathogenesis of HCC. Increasing evidence has suggested the inhibition of these two cyclooxygenase isoforms as an effective preventative as well as therapeutic strategy against HCC.8−10 As a key metabolic enzyme, NNMT is involved in tumorigenesis by altering the epigenetic state of cancer cells.11,12 Activation of DLD in the Krebs cycle may decelerate the glycolytic switch, thus attenuating active cell proliferation. This enzyme also functions as a constitutive source of reactive oxygen species (ROS) in mitochondria and results in apoptosis.13 Overexpression of TSPAN8 has been reported in liver cancer, where it induces tumor angiogenesis and promotes metastasis via regulation of 3154



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Scheme 2. Stereochemical Relationships of (R, R)-1, (S, S)-1, (R, S)-1, and (S, R)-1

Scheme 3. Proposed Mechanism for the Formation of (R,R)-1 and (R,R)-2 from (R)-3

of Chinese Medicine. A voucher specimen (2012-10-018) was deposited in our laboratory. Extraction and Isolation. The air-dried and powdered plant material (6 kg) was extracted exhaustively with 95% EtOH (3 × 60 L) under reflux. The extract was suspended in H2O (2 L) and successively extracted with petroleum ether (60−90 °C; 3 × 2 L), EtOAc (3 × 2 L), and n-BuOH (3 × 2 L). The n-BuOH extract (36 g) was subjected to CC (SiO2, 200−300 mesh) with CHCl3/MeOH (95:5 to 0:100, v/ v) to afford four fractions, A−D. Fr. C (3 g) was subjected to column chromatography (CC) (ODS, MeOH/H2O, 50:50 → 90:10; then Sephadex LH-20, MeOH) to afford 3 (21 mg), and 4 (6 mg). Fr. D (1.5 g) was repeatedly separated by CC (ODS, MeOH/H2O, 50:50 → 100:0, v/v) to give a subfraction that mainly contained compounds 1

and 2. The mixture was further separated by semipreparative HPLC with MeOH/0.1% HCOOH−H2O (1:1) to yield 1 (4.5 mg, retention time 8.6 min) and 2 (3.6 mg, retention time 11.0 min). The analysis of the stereoisomers was performed using HPLC on a Daicel Chiralpak OZ-H column (4.6 mm × 250 mm, 5 μm; n-hexane/0.1% CH3COOH−EtOH, 76:24; flow rate 1.0 mL/min). Diselaginellin A (1): red powder; [α]25D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 270 (4.30), 300 (4.26), 420 (4.00) nm; ECD (MeOH), no Cotton effects; IR (KBr) 3414, 2924, 2200, 1596, 1513, 834 cm−1; for 1H and 13C NMR data, see Table 1; HRESIMS 1007.32195 [M + H ]+ (calcd for C68H47O9+, 1007.32147). Diselaginellin B (2): red powder; [α]25D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 285 (4.32), 300 (4.30), 430 (3.96) nm; ECD 3155



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Figure 3. Effects of diselaginellin B (2) on apoptosis induction, cell cycle, and metastasis inhibition in SMMC-7721 cells. (A, B) Flow cytometric analysis of annexin V/PI-positive cells was performed to evaluate apoptotic cells. (C) The percentages of G1, S, and G2 cell populations were analyzed by flow cytometry, means ± SD (n = 3). (D) Cell invasion and migration were measured by a Matrigel invasion assay and a Transwell assay. Cells were treated with the indicated concentrations (0.5, 1, and 2 μM) of 2 for 48 h. After staining with crystal violet, invaded or migrated cells were visualized and scored by fluorescence microscopy, with paclitaxel (100 nM) as the positive control. (E) The values for the Transwell invasion and migration assays are the means ± SD (n = 3; *p < 0.05, **p < 0.01). MTT Assay. Briefly, 5 × 104 cells were seeded into 96-well plates and cultured overnight. Then cells were treated with the indicated concentrations of the isolates for 48 h. After 10 μL of MTT (5 mg/ mL) was added for 4 h, the formazan product was dissolved in DMSO. The absorbance was measured at 490 nm by a microplate reader. Cell Cycle Analysis. SMMC-7721 cells were treated with compound 2 at 2.5 or 5 μM for 48 h, washed with PBS, and fixed with 70% ethanol at 4 °C overnight. Cells were collected by

(MeOH), no Cotton effect; IR (KBr) 3397, 2923, 2852, 2192, 1596, 1496, 830 cm−1; for 1H and 13C NMR data, see Table 1; HRESIMS 1007.32122 [M + H ]+ (calcd for C68H47O9+, 1007.32146). Cell Culture. The cell line SMMC-7721 was obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, People’s Republic of China). Cells were grown in RPMI 1640 (Gibco) with 10% fetal bovine serum and cultivated at 37 °C with 5% CO2. 3156



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Figure 4. Effects of diselaginellin B (2) on the gene expression profile in SMMC-7721 cells. (A) Heat map showing the hierarchical clustering analysis of differentially expressed genes between the control group and the diselaginellin B (2) incubation group (8 μM for 3 days), n = 3. Each row corresponds to one mRNA, red: up-regulated, green: down-regulated. (B) SMCC-7721 cells were exposed to diselaginellin B (2) (8 μM). The mRNA levels of the metabolism- and metastasis-related genes were determined by RT-qPCR, with actin as an internal control; means ± SD (n = 3; **p < 0.01). centrifugation, washed, and incubated with RNase (0.1 mL) at 37 °C for 30 min followed by staining with propidium iodide (PI) (400 μL) for 30 min in the dark. The cell cycle phase distribution was analyzed using a FACSCalibur flow cytometer (BD Biosciences, CA, USA). Annexin V-FITC/Propidium Iodide Double Staining. SMMC7721 cells were exposed to compound 2 at 2.5, 5, or 10 μM for 48 h. The cells were washed, resuspended with binding buffer (5 × 105 cells/mL), and stained with annexin V-FITC and PI (5 μL) for 10 min at room temperature in the dark. The samples were immediately analyzed by flow cytometry (BD Biosciences, CA, USA). Transwell Migration and Invasion Assay. Cell migration and invasion were assessed in Transwell chambers (Corning, NY, USA). For the migration assay, 2 × 104 cells were seeded onto the upper surface of a Transwell filter (8 μm pore size) in serum-free RPMI and incubated with the indicated concentrations of compound 2 for 48 h. A volume of 600 μL of 10% fetal bovine serum medium was added in the bottom chamber. After incubation, the cells on the upper surface of the membrane were removed using cotton swabs. The cells that migrated to the underside of the membrane were stained with crystal violet and scored using a fluorescence microscope in five random fields. For the Transwell invasion assay, the upper filter membranes were coated with matrigel (BD Biosciences, Bedford, MA, USA) for 2 h at 37 °C. Microarray Analysis. Microarray analysis of both the diselaginellin B (2)-treated group and the control group was performed in triplicate. The total RNA was extracted from 1 × 107 cells using TRIzol Reagent (Life Technologies) and labeled. The labeled samples were hybridized to the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix), which contained 38 sets of 500 human genome probes. After DNA hybridization, the Affymetrix GeneChip Command Console 3.2 (AGCC) software was used to extract data. Statistical Analyses. The data represent the mean ± SD from three independent experiments. Two-tailed Student’s t tests and oneway analysis of variance (ANOVA) were used to evaluate the data. The difference was considered to be statistically significant at *p < 0.05 and **p < 0.01.

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Copies of 1D and 2D NMR spectra of compounds 1 and 2; tables of differentially expressed genes in SMMC-7721 treated with diselaginellin B (2) and primer-probe sets for RT-PCR validation of microarray data (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-25-85811916. E-mail: [email protected]. ORCID

Yuan Cao: 0000-0002-0019-3709 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Dr. C. Guenat (Novartis Pharma AG) for recording the HRESI mass spectra. This project was supported financially by the National Natural Science Foundation of China (No. 81102772), Jiangsu Planned Projects for Postdoctoral Research Funds (No.1302083B), and Jiangsu Government Scholarship for Overseas Studies (JS2014-133).

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REFERENCES

(1) Siegel, R.; Naishadham, D.; Jemal, A. Ca-Cancer J. Clin. 2012, 62, 10−29. (2) Tang, Z. Y.; Ye, S. L.; Liu, Y. K.; Qin, L. X.; Sun, H. C.; Ye, Q. H.; Wang, L.; Zhou, J.; Qiu, S. J.; Li, Y.; Ji, X. N.; Liu, H.; Xia, J. L.; Wu, Z. Q.; Fan, J.; Ma, Z. C.; Zhou, X. D.; Lin, Z. Y.; Liu, K. D. J. Cancer Res. Clin. Oncol. 2004, 130, 187−196. (3) Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China; People’s Medical Publishing House: Beijing, 2015; Vol 1, pp 226−227. (4) Editorial Board of “Zhong Hua Ben Cao”, State Administration of Traditional Chinese Medicine of the People’s Republic of China. Chinese Materia Medica (Zhong Hua Ben Cao); Shanghai Scientific and Technical Publishers: Shanghai, 1999; Vol 4, p 387. (5) (a) Zhang, L. P.; Liang, Y. M.; Wei, X. C.; Cheng, D. L. J. Org. Chem. 2007, 72, 3921−3924. (b) Cheng, X. L.; Ma, S. C.; Yu, J. D.; Yang, S. Y.; Xiao, X. Y.; Hu, J. Y.; Lu, Y.; Shaw, P. C.; But, P. P. H.;

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00404. 3157



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Diselaginellin B, an Unusual Dimeric Molecule from Selaginella

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