Structure Elucidation and Biological Activity of Two New

Article pubs.acs.org/JAFC

Structure Elucidation and Biological Activity of Two New Trichothecenes from an Endophyte, Myrothecium roridum Ting Lin, Guanghui Wang, Yuqi Zhou, Dequan Zeng, Xiaoxuan Liu, Rong Ding, Xin Jiang, Dan Zhu, Wenjun Shan, and Haifeng Chen* School of Pharmaceutical Sciences, Xiamen University, South Xiangan Road, Xiamen, Fujian 361102, China S Supporting Information *

ABSTRACT: Worldwide, many different grains are infected by various fungi that may produce trichothecene mycotoxins. Fungi that produce trichothecenes, as well as the trichothecenes themselves, are potential problems for public health. On the other hand, trichothecenes possess multiple biological activities. Reduced toxicity may result in their applications in the pharmaceutical field. Two new trichothecenes along with seven known trichothecenes were isolated from an endophyte of the herb plant Ajuga decumbens. Their structures were deduced from 1D and 2D NMR data. The results of MTT assays revealed that new trichothecene 2′,3′-epoxymyrothecine A, 1, and myrothecine A, 3, exhibited much lower toxicity compared to other trichothecenes. New trichothecene 2′,3′-epoxymyrothecine A, 1, could induce phosphorylation of JNK (c-Jun N-terminal protein kinase) protein and the PARP (poly ADP-ribose polymerase) cleavage, and eventually induce apoptosis in cancer cells. These results point out the possibility for application of trichothecenes as chemotherapeutic agent. KEYWORDS: trichothecenes, endophyte, mycotoxin, chemotherapeutic agent

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INTRODUCTION Trichothecenes are a large family of chemically related mycotoxins produced by various species of Fusarium, Myrothecium, Trichoderma, Trichothecium, Cephalosporium, Verticimonosporium, and Stachybotrys.1 These mycotoxins are produced in Fusarium-infested grains such as corn, wheat, and barley, and ingestion of contaminated grain can result in a variety of symptoms including diarrhea, hemorrhaging, and feed refusal.2 Potentially hazardous concentrations of the trichothecene mycotoxins can occur naturally in moldy grains, cereals, and agricultural products.3 They can be found worldwide in cereal grains and in animal feed and human food produced from contaminated grains, which creates a food safety risk.1 Trichothecenes can enter human and animal food chains through breakfast cereals, bakery products, snack foods, beer, and compound feeds made from small grains and maize.4 These mycotoxins are characterized by a tricyclic 12,13-epoxytrichothec-9-ene (trichothecene) ring structure. They can be classified into two different types: simple or nonmacrocyclic trichothecenes, and macrocyclic trichothecenes. The simple or nonmacrocyclic trichothecenes can be divided further into two subgroups based on the presence or absence of a carbonyl function at C-8.5 Type B trichothecenes have a keto function at C-8, while type A trichothecenes have a hydrogen, hydroxyl, or ester function at C-8.2 Simple trichothecenes including type A and type B have variations at four sites (C-3, C-4, C-7, and C15) and are subdivided into epoxy types and de-epoxy types, which are structural analogues bearing an ethylene group, which has not been epoxidized, and they may be biosynthetic intermediates on the pathway to corresponding epoxidized derivatives.6 Type A trichothecenes are generally more toxic to animals. The naturally occurring macrocyclic trichothecenes are classified as verrucarins (mainly C27 compounds) and roridins (mainly C29 metabolites). They are characterized by a family of © 2014 American Chemical Society

sesquiterpenols with a diacyl residue anchored at the 4β and 15 positions. Among these trichothecenes, a number have been demonstrated to play a substantial role in malaria therapy,7 immunomodulation,8 and cancer prevention.9 The inhibitory effect of trichothecene mycotoxins on rapidly dividing cells was the basis for their evaluation as antitumor chemotherapy drugs during the late 1970s and early 1980s.10 They are cytotoxic to most eukaryotic cells and inhibit protein synthesis in a variety of eukaryotic cells.11 Higher efficacy and lower toxicity trichothecenes might have significant potential for use as a chemotherapeutic agent in cancer treatment. Because of their multiple functions, the trichothecene mycotoxins have become a frequent research subject in the fields of medicine, agriculture, animal breeding, food safety, and toxicology.12 From an endophyte of the herb Ajuga decumbens, two new and seven known trichothecenes were isolated. Their structures were elucidated by spectroscopic methods, and cytotoxicity structure−activity relationships were investigated.

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MATERIALS AND METHODS

General Experimental Procedures. NMR spectra were taken on a Avance III-600 NMR spectrometer (Bruker BioSpin International AG, Zurich, Switzerland) with TMS as an internal standard. HR-FTMS data were acquired using an Apex Ultra 7.0 FT-MS (Bruker Corp., Billerica, MA, USA). Optical rotations were obtained on a PerkinElmer 341 automatic polarimeter (Waltham, MA, USA) with CHCl3 as the solvent. IR spectra were recorded on a Nicolet Avatar 330 FT IR spectrometer (Thermo Electron Corp., Madison, WI, USA). Sephadex Received: Revised: Accepted: Published: 5993

January 14, 2014 June 5, 2014 June 8, 2014 June 8, 2014 dx.doi.org/10.1021/jf501724a | J. Agric. Food Chem. 2014, 62, 5993−6000

Journal of Agricultural and Food Chemistry

Article

LH-20 (40−70 μm) (Amersham Pharmacia Biotech AB, Uppsala, Sweden), silica gel (200−300 mesh) (Qingdao Marine Chemical, Inc., Qingdao, China), and Lichroprep reversed-phase RP-18 silica gel (40− 63 μm) (Merck, Darmstadt, Germany) were used for column chromatography. TLC was carried out using glass-precoated silica gel GF254 (Qingdao Marine Chemical, Inc., Qingdao, China) and visualized under UV light or by spraying with vanillin ethanol reagent, which contains H2SO4. The cell culture medium, Dulbecco’s modified Eagle’s medium (DMEM; high glucose, without sodium pyruvate, HyClone), and fetal bovine serum (FBS, HyClone) were purchased from HyClone Laboratories Inc. (Logan, UT, USA). Acrylamide/bisacrylamide solution (30%; 29:1) was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Protein content was tested using a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Antipoly(ADP-ribose) polymerase-1 (PARP-1), anti-β-actin mouse antibody, horseradish peroxidase (HRP)-labeled anti-rabbit/mouse IgG (H + L), and dimethyl sulfoxide (DMSO) were obtained from SigmaAldrich Corp. (St. Louis, MO, USA). Cytochrome c was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). DAPI (4′,6′-diamidino-2-phenylindole) was purchased from Pierce (Rockford, IL, USA). Fungal Material. The fungus was isolated from current-year root of Ajuga decumbens collected from Fuzhou, Fujian, China. A BLAST search result showed that the internal transcribed spaces (ITS) sequence of this fungus was highly homologous (100% percent similarity) to that of Myrothecium roridum (E-1069), indicating that this fungus belongs to this genus. Fermentation and Extraction. The strain was cultivated at 28 °C with 5 L of PDA (potato dextrose agar) media for 16 d. After fermentation, the culture medium was chopped and extracted with ethyl acetate/methanol (80:20) at room temperature overnight. The combined organic layer was concentrated under vacuum until 4.1 g of residue was obtained. Isolation and Spectral Data. The crude extract (4.1 g) was separated into 18 fractions (fraction 1 to fraction 18) by column chromatography on RP-18 silica gel (100 g, 36 mm × 230 mm), eluted by methanol/H2O (30:70, 50:50, 70:30, and 100:0, 1 L for each gradient, 100 mL per collection bottle). Fraction 1 (0.2315 g, fraction volume: 200 mL); fraction 2 (0.0380 g, fraction volume: 100 mL); fraction 3 (0.0611 g, fraction volume: 300 mL); fraction 4 (0.1080 g, fraction volume: 400 mL); fraction 5 (0.0431 g, fraction volume: 100 mL); fraction 6 (0.0836 g, fraction volume: 200 mL); fraction 7 (0.0386 g, fraction volume: 100 mL); fraction 8 (0.1195 g, fraction volume: 300 mL); fraction 9 (0.0674 g, fraction volume: 100 mL); fraction 10 (1.4100 g, fraction volume: 200 mL); fraction 11 (0.0745 g, fraction volume: 200 mL); fraction 12 (0.1649 g, fraction volume: 100 mL); fraction 13 (0.0476 g, fraction volume: 100 mL); fraction 14 (0.1802 g, fraction volume: 300 mL); fraction 15 (0.6822 g, fraction volume: 100 mL); fraction 16 (0.7478 g, fraction volume: 100 mL); fraction 17 (0.1352 g, fraction volume: 100 mL); and fraction 18 (0. 0390 g, fraction volume: 400 mL). Fraction 4 (108 mg) was separated using the Sephadex LH-20 (elution with 100% methanol, 25 mm × 1000 mm, 100 g, 400 mL) to give three subfractions (fraction 4-a to fraction 4-c). Fraction 4-a (0.0784 g, fraction volume: 15 mL) was separated by column chromatography on silica gel (2.35 g, 10 mm × 250 mm, elution with chloroform, 600 mL) to afford compound 1 (19.9 mg). Fraction 6 (83.6 mg) was chromatographed on normal phase silica gel (2.51 g, 10 mm × 250 mm) and eluted with chloroform (120 mL) to yield compound 3 (9.0 mg, fraction volume: 30 mL). Fraction 9 (67.4 mg) was purified using the Sephadex LH-20 (25 mm × 1000 mm, 100 g) column, eluted with methanol (300 mL), and two subfractions (fraction 9-a, 39.6 mg, and fraction 9-b, 20.4 mg) were obtained. Compound 9 (4.9 mg) was obtained from fraction 9-a eluted with petroleum ether−EtOAc [5:1(60 mL), 4:1 (100 mL), 3:1 (80 mL), 2:1 (60 mL), 1.5:1 (150 mL), 1:1 (100 mL), v/v] on column chromatography over normal phase silica gel (1.5 g, 10 mm × 250 mm). Fraction 10 (1.41 g) was subjected to the Sephadex LH-20 (180 g) (25 mm × 1800 mm, elution with 100% methanol, 800 mL), and two

fractions (fraction 10-a, 36.6 mg, and fraction 10-b, 841 mg) were collected. Fraction 10-b (841 mg) was fractionated on a silica gel column (8.4 g, 20 mm × 350 mm) eluted with petroleum ether− EtOAc of increasing polarity [4:1 (200 mL), 3:1 (240 mL), 2.5:1 (280 mL), 2:1 (90 mL), 1:1 (200 mL), v/v] to give compound 5 (4 mg, fraction volume: 10 mL), compound 6 (25.7 mg, fraction volume: 20 mL), and compound 2 (3.9 mg, fraction volume: 60 mL). Fraction 11 (74.5 mg) was chromatographed on the Sephadex LH20 (25 mm × 1000 mm, 100 g) and eluted with methanol (400 mL). Two fractions (fraction 11-a, 26 mg, and fraction 11-b, 43 mg) were made. Fraction 11-a (26 mg) was isolated with column chromatography over normal-phase silica gel [1.0 g, 8 mm × 250 mm, eluted with chloroform−methanol, v/v, 200:1 (100.5 mL)] to afford comopund 4 (6.8 mg). Fraction 13 (47.6 mg) was purified by normal phase silica gel column chromatography (1.5 g, 10 mm × 250 mm), eluted with petroleum ether−chloroform [4:1 (100 mL), 3:1 (80 mL), 2:1 (90 mL), 1:1 (100 mL), 1:2 (80 mL), v/v], and compound 7 (4.1 mg, fraction volume: 35 mL) was obtained. Fraction 14 (180.2 mg) was further subjected to the Sephadex LH20 (180 g, 25 mm × 1800 mm) column, eluted with methanol (600 mL). Subfraction 14-a (15.3 mg, fraction volume: 20 mL) was purified using a normal phase silica gel column [0.5 g, 8 mm × 250 mm, eluted with chloroform−methanol (100:1, v/v, 400 mL)] to yield compound 8 (3.0 mg, fraction volume: 10 mL). 2′,3′-Epoxymyrothecine A, 1: white powder; [α]20 D +12.8 (c 0.525, chloroform); IR (KBr) νmax 3428, 2920, 2850, 1745, 1711, 1461, 1383, 1181, 1098, 1058, 1023, 754 cm−1; 1H and 13C NMR data shown in Table 1; HR-FT-MS, m/z 541.2045 [M + Na]+ (calculated for C27H34O10Na, 541.2146). 13′,14′-Hydroxymytoxin B, 2: white powder; [α]20 D +60 (c 0.1, chloroform); IR (KBr) νmax 3348, 2921, 2851, 1720, 1712, 1658, 1461, 1441, 1383, 1085, 1180 cm−1; 1H and 13C NMR data shown in Table 1; HR-FT-MS, m/z 569.2361 [M + Na]+ (calculated for C29H38O10Na, 569.2465). Cleaved-PARP and JNK Protein Phosphorylation Western Blotting. HepG2 (hepatocellular carcinoma, ATCC) cells were exposed to 5-Fu (5-fluorouracil) (20 and 50 μM) and compounds 1−9 at concentrations of 1 (50 μM), 2 (50 nM), 3 (50 μM), 4 (5 nM), 5 (50 nM), 6 (50 nM), 7 (50 nM), 8 (50 nM) and 9 (1 μM) for 8 h. HepG2, A549 (human lung adenocarcinoma, ATCC) and MCF-7 (human breast adenocarcinoma, ATCC) cells were exposed to 2′,3′epoxymyrothecine A, 1, at concentrations of 0, 20, 50, and 100 μM for 8 h. HepG2 cells were treated with 20 μM 1 for different periods of time (0, 2, 4, and 8 h) respectively. After the treatment, cells were lysed with prepared lysis buffer with a WH-3 vortex machine (Shanghai Qingpu-Huxi Instruments Factory, Shanghai, China). For Western blot analysis, an amount of 25 μL of the protein sample containing 40 μg of protein was separated by 8% SDS−polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The level of protein expression was determined using specific primary antibodies, PARP and β-actin, followed by rabbit/mouse HRP-conjugated secondary antibodies. Cytotoxicity Assay. In vitro cytotoxicity of isolated compounds was evaluated by an MTT assay. Briefly, four different cancer cells [HepG2, MCF-7, A549, and SMMC-7721 (human hepatocellular carcinoma, Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China)] were plated at 3000 cells/well in a 96-well plate and treated with the different concentrations of the isolated compounds for 48 h. Three independent experiments were performed and the results shown as the percentage of cell inhibition ratio. Immunofluorescence Analysis of Cytochrome c. HepG2 cells were seeded onto glass coverslips and treated with compounds for 12 h. Cells were fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. The cells were washed twice with PBS and blocked with 5% BSA in PBS for 30 min at room temperature. After washing, the cells were incubated with anti-cytochrome c (diluted 1:500 in PBS + 1% BSA) overnight at 4 °C. The cells were then washed twice in PBS 5994

dx.doi.org/10.1021/jf501724a | J. Agric. Food Chem. 2014, 62, 5993−6000


Article

Table 1. 1H NMR (600 MHz) and 13C NMR (125 MHz) Data for Compounds 1 and 2 in CDCl3 1 δH, multi (J in Hz) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′

4.01, 1.83, 1.56, 5.43,

d (4.3) m m dd (7.9, 2.6)

2.11, 1.47, 1.40, 1.82,

m dd (5.4, 14.0) m m

2.16, m 3.56, s 2.23, 1.88, 1.16, 4.21,

m m s m

1.21, s 3.53, s 2.75, 1.71, 4.24, 4.10,

dt (5.6, 13.2) dt (5.0, 14.4) m m

4.61, dd (2.3, 10.6) 4.16, dt (2.8, 11.6) 2.51, ddd (2.3, 9.4, 14.4) 6.62, m 5.96, dd (1.8, 11.4) 3.57, s

histograms were further analyzed through the WinMDI 2.9 software program (Scripps Institute, La Jolla, CA, USA).

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2 δC

δH, multi (J in Hz)

δC

80.8 d 28.4 t

3.86, m 2.49, dd (15.7, 8.1) 2.11, m 5.80, dd (8.1, 3.5)

79.1 d 35.0 t

77.9 50.8 43.8 27.6

d s s t

30.4 t 72.6 43.9 69.3 76.6 39.9

s d d s t

9.7 q 73.7 t 26.3 q 167.6 s 56.3 d 65.3 s 22.9 t 66.9 t 151.3 s 108.5 d 23.2 t 147.1 d 121.9 d 164.9 s 74.0 d

4.65 1.92, m 2.05, m 1.92, m 5.45, d (4.6) 3.62, d (5.2) 3.14, 2.82, 0.81, 4.33, 4.03, 1.73,

d (4.0) m s m m s

5.74, s 3.71, 2.81, 4.10, 4.08,

d (13.2) d (4.2) m m

2.86, m 1.96, m 2.20, m 6.49, m 5.45, d (4.6) 3.85, s 4.06, d (2.6) 3.95, s

73.7 43.9 42.9 20.6

RESULTS AND DISCUSSION Structure Elucidation. 2′,3′-Epoxymyrothecine A, 1, was isolated as a white powder. The molecular formular was established as C27H34O10. The 1H and 13C NMR spectra of 1 were similar to those of myrothecine A,13 except for the presence of signals [δH 3.53 (1H, S, H-2′), δC 56.3 (C-2′), δC 65.3 (C-3′), δH 4.61 (1H, dd, J = 2.3, 10.6 Hz, H-7′), δC 108.5 (C-7′), δC 151.3 (C-6′)] and the absence of signals [δH 5.78 (1H, brs, H-2′), δC 117.3 (C-2′), δC 154.1 (C-3′), δH 1.96, 1.65 (2H, m, H-7′), δC 29.5 (C-7′), δC 87.3 (C-6′), δC 213.0 (C13′), δH 2.30 (1H, s, H-14′), δC 28.7 (C-14′)] observed in the NMR spectra of myrothecine A due to the epoxidation between C-2′ and C-3′ in myrothecine A, 3, the double bond of C-6′ and C-7′, and the absence of an acetyl group that was connected to C-6′ in myrothecine A. The HMBC correlations from proton δH 4.21 (2H, m, H-15) to C-1′ (δC 167.6), C-5 (δC 50.8), and C-7 (δC 27.6) revealed the linkage between C-15 and C-1′ via an oxygen bridge. The HMBC correlations from proton δH 4.01 (1H, d, J = 4.3 Hz, H-2) to C-13 (δC 39.9), C-5 (δC 50.8), and C-11 (δC 69.3) revealed the oxygen bridge between C-2 and C-11. The HMBC correlations from proton δH 5.43 (1H, dd, J = 2.6, 7.9 Hz, H-4) to C-12 (δC 76.6), C-2 (δC 80.8), and C-11′ (δC 164.9) revealed the ester linkage between C-4 and C-11′. The HMBC correlations from proton δH 4.24 (2H, m, H-5′) to C-3′ (δC 65.3) and C-6′ (δC 151.3) revealed the oxygen bridge linkage between C-5′ (δC 66.9) and C-6′. Other key HMBC correlations are shown in Figure 2. Therefore, the planar structure of compound 1 was determined. Moreover, the relative configuration of 1 was given by its nuclear Overhauser effect spectroscopy (NOESY) NMR spectrum which displayed the cross peaks of δH 4.01 (1H, d, J = 4.3 Hz, H-2) with δH 5.43 (1H, dd, J = 2.6, 7.9 Hz, H-4), δH 4.01 (1H, d, J = 4.3 Hz, H-2) with δH 2.21 (1H, m, H-10), δH 3.56 (1H, s, H-11) with δH 2.21 (1H, m, H-10), and δH 1.25 (3H, s, H-16) with δH 1.16 (3H, s, H-14). 13′,14′-Hydroxymytoxin B (2) was isolated as a white powder. The molecular weight and formula (C29H38O10) were determined from the HR-FT mass spectrum (C29H38O10Na) and the 13C NMR data. The 1H and 13C NMR spectra of 2 were nearly identical to those of 14′-hydroxymytoxin B (5),5 except for the presence of signals [δH 4.06 (1H, d, J = 2.6 Hz, H-13′), δC 75.8 (C-13′)] ascribable to a hydroxymethylene group and the absence of a signal at δC 214.1 (C-13′), observed in the NMR spectra of 14′-hydroxymytoxin B due to the 13′ carbonyl group. HMBC correlations from proton δH 4.33 (1H, m, H-15) to C-7 (δC 20.9), C-6 (δC 42.9), C-11′ (δC 68.7), and C-1′ (δC 166.4) revealed the ester linkage between C-15 and C1′. The HMBC correlations from proton δH 3.16 (1H, m, H-2) to C-13 (δC 79.1) and C-11 (δC 67.9) revealed the oxygen bridge between C-2 and C-11. The HMBC correlations from proton δH 5.80 (1H, dd, J = 3.5, 8.1 Hz, H-4) to C-12 (δC 65.6), C-2 (δC 47.6), and C-11′ (δC 165.8) revealed the ester linkage between C-4 and C-11′. Other key HMBC correlations are shown in Figure 2. Therefore, the structure of 2 was deduced to be the 13′-hydroxy derivative of 14′-hydroxymytoxin B. The relative configurations of 2 were determined by the analysis of the NOESY spectrum which displayed the nuclear Overhauser effect (NOE) correlations of δH 5.45 (1H, d, J = 4.6 Hz, H-10) with δH 3.62 (1H, d, J = 5.2 Hz, H-11), δH 5.45 (1H, d, J = 4.6 Hz, H-10) with δH 1.73 (3H, s, H-16), δH 5.74 (1H, s,

d s s t

27.6 t 140.4 s 118.8 d 67.9 d 65.6 s 47.6 t 7.7 q 64.3 t 23.3 q 166.4 s 117.0 d 155.1 s 25.7 t 63.9 t 78.6 s 21.8 t 26.6 t 149.2 d 118.8 d 165.8 s 77.6 d 75.8 d 62.1 t

and detected by anti-mouse IgG conjugated with Cy3 (1:1000) at room temperature for 30 min. Cells were costained with DAPI to visualize nuclei. The images were taken using LSM-510 confocal laser scanning microscope system (Carl Zeiss, Oberkochen, Germany). To investigate whether these compounds may induce the mitochondrial release of cytochrome c, we performed immunofluorescence analysis of cytochrome c in HepG2 cells treated with 5-Fu (50 μM), 1 (50 μM), 3 (50 μM), 5 (50 nM), 6 (50 nM), and 8 (50 nM) for 12 h, and observed that they can induce a diffuse cytosolic staining of cytochrome c. Flow Cytometry for Cell Cycle Distribution. HepG2 cells (50% confluent) were synchronized by serum starvation for 24 h and treated with 5-Fu (50 μM), compound 1 (50 μM), 2 (50 nM), 3 (50 μM), 4 (5 nM), 5−8 (50 nM), and 9 (1 μM) in complete medium for 12 h. The cells were washed with cold PBS and centrifuged at 1000 rpm for 5 min. 70% ethanol was employed to fix the cells overnight at 4 °C. The cells were incubated using PBS with RNase (10 μg/mL) (Tiangen, Beijing, China) for 30 min, stained with propidium iodide (50 μg/mL) at room temperature for 30 min, and determined by FAC Plus flow cytometry (Beckman Coulter, Miami, FL, USA). The DNA 5995



Article

Figure 1. Compounds isolated from M. roridum: 1, 2′,3′-epoxymyrothecine A; 2, 13′,14′-hydroxymytoxin B; 3, myrothecine A; 4, myrotoxin A; 5, 14′-hydroxymytoxin B; 6, vertisporin; 7, mytoxin C; 8, roridin E; 9, 12′-hydroxyroridin E.

H-2′) with δH 0.81 (3H, s, H-14), δH 5.74 (1H, s, H-2′) with δH 3.85 (1H, s, H-12′), and δH 0.81 (3H, s, H-14) with δH 3.62 (1H, d, J = 5.2 Hz, H-11). Two new trichothecenes, 2′,3′-epoxymyrothecine A, 1, and 13′,14′-hydroxymytoxin B, 2, along with seven known trichothecenes, myrothecine A, 3,12 myrotoxin A, 4,13 14′hydroxymytoxin B, 5,5 vertisporin, 6,14 mytoxin C, 7,15 roridin E, 8,16 and 12′-hydroxyroridin E, 9,17 were isolated from an endophyte of the herb Ajuga decumbens; their structures are shown in Figure 1. Since Fusarium and other related fungi that produce trichothecenes infect various agricultural products, especially important crops, they have been associated with intoxication of humans and animals all over the world. Trichothecenes are a well-known major class of mycotoxins. Many new macrocyclic trichothecenes have been isolated from different fungi possessing cytotoxic,9 immunomodulation,18 antimicrobial,19 and phytotoxic20 properties. MTT assays were used to evaluate these trichothecenes’ toxicity. 2′,3′-Epoxymyrothecine A, 1, and myrothecine A, 3, exhibited more than 1000-fold less toxicity than other trichothecenes (Table 2). The 12,13-epoxy ring is the most important structural feature leading to the remarkable differences in toxicity. A promising target for preharvest detoxification is the epoxide group located at C-12. 2′,3′Epoxymyrothecine A, 1, and myrothecine A, 3, do not have an epoxy ring at C-12 and C-13. This variation can lead to

Figure 2. Key HMBC and NOE correlations of 1 and 2.

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programmed cell death (apoptosis).26 Cell cycle arrest experiments were carried out. Nine trichothecenes could induce the G1 cell cycle arrest in HepG2 cells (Figure 5). 2′,3′-Epoxymyrothecine A, 1, and myrotoxin A, 4, exhibited significant effects of G1 cell cycle arrest in HepG2, nearly 20% greater than the control (Figure 5). In structure comparions of these two trichothecenes to other trichothecenes, the most obvious difference was the substitution at C-6′. There was no substituent at C-6′ in these two trichothecenes (2′,3′epoxymyrothecine A and myrotoxin A), whereas the other trichothecenes had a substitution group at C-6′. Therefore, this difference should be important for the G1 cell cycle arrest activity of trichothecenes. Cells are continuously exposed to a variety of environmental stresses and have to decide “to live or die” depending on the types and strength of stress. Among the many signaling pathways that respond to stress, mitogen-activated protein kinase (MAPK) family members are crucial for the maintenance of cells.27 Three subfamilies of MAPKs have been identified: c-Jun N-terminal kinases (JNKs), extracellular signal-regulated kinases (ERKs), and p38-MAPKs. It has been shown that ERKs are important for cell survival, whereas JNKs were deemed stress-responsive and thus involved in apoptosis.27 JNK was discovered almost 20 years ago as the protein kinase responsible for phosphorylating c-Jun at Ser-63 and Ser73.28 JNKs belong to the family of mitogen-activated protein kinases (MAPK) that are responsive to a variety of environmental stimuli.29,30 JNKs (also known as stress-activated protein kinases; SAPKs) are ubiquitously expressed, and the JNK stress pathways participate in many different intracellular signaling pathways that control a spectrum of cellular processes, including cell growth, differentiation, transformation, or apoptosis.31−37 Transient JNK activation promotes cell survival, while prolonged JNK activation induces cellular apoptosis.38 In order to explain the structure−activity relationships of these trichothecenes, the PARP-cleavage and phosphorylation of JNK protein activities were carried out in HepG2 cells. With the exception of trichothecenes 2, 4, 7, and 9, all of the other trichothecenes could induce PARP-cleavage in HepG2 cells (Figure 3). Also, with the exception of trichothecenes 2 and 9, all of the other trichothecenes could induce phosphorylation of JNK protein in HepG2 cells (Figure 3). Trichothecenes 1 and 3, in which the epoxy ring typically located at C-12 and C-13 was absent and the olefinic bond, which is typically located at C-9 and C-10, was substituted with a hydroxy group, could induce PARP-cleavage and phosphorylation of JNK protein in HepG2 cells (Figure 3). These results indicated that the epoxy ring at C-12 and hydroxy substitution at C-9 were important for PARP-cleavage and phosphorylation of JNK protein in HepG2 cells. This conclusion can be further confirmed by the comparison of 1 and 4. The epoxy ring at C-12 of 4 was open in 1, and the olefinic bond at C-9 of 4 was substituted with a hydroxy group in 1. For trichothecenes 1, 4, and 7, the epoxy ring at C-12 and the hydroxy substitution at C-9 were absent in trichothecenes 4 and 7. Among these three trichothecenes, the epoxy ring located at C-2′ and C-3′ did not play an important role in PARP-cleavage and phosphorylation of the JNK protein in HepG2 cells (Figure 3). For trichothecenes 2, 5, and 6, the substituent group located at C-6′ was the major cause for the activities in HepG2 cells. In other two trichothecenes, 2 and 5, no ring system existed at C-6′. In a comparison of these two trichothecenes, the only difference between them was that the

Table 2. IC50 Values of Compounds (1−9) against Four Different Cancer Cell Lines (7721, A549, HepG2, MCF-7) 1 2 3 4 5 6 7 8 9

A549

MCF-7

HepG2

7721

36.45 ± 2.79 μM 53 ± 3.36 nM 95 ± 3.51 μM 3.56 ± 1.23 nM 49 ± 1.26 nM 47 ± 3.30 nM 33 ± 1.86 nM 55 ± 3.33 nM 2.08 ± 0.71 μM




different trichothecene structures that result in differences in toxicity. 2′,3′-Epoxymyrothecine A, 1, and myrothecine A, 3, exhibited much lower toxicity compared to those trichothecenes which possess a 12,13-epoxy ring (Table 2). PARP-1 is a nuclear protein that has important roles in the maintenance of genomic integrity.21 Cleavage of PARP-1 by caspases is considered to be a hallmark of apoptosis.22,23 The PARPcleavage activity of nine trichothecenes in HepG2 cells was tested (Figure 3). With the exception of trichothecenes 2, 4, 7,

Figure 3. PARP-cleavage and phosphorylation of JNK protein in treated HepG2 cells. Cells were treated with 5-Fu (20 and 50 μM), 1 (50 μM), 2 (50 nM), 3 (50 μM), 4 (5 nM), 5-8 (50 nM), and 9 (1 μM) for 8 h.

and 9, the other trichothecenes could induce PARP-cleavage in HepG2 cells (Figure 3). The results revealed that 2′,3′epoxymyrothecine A, 1, myrothecine A, 3, 14′-hydroxymytoxin B, 5, vertisporin, 6, and roridin E, 8, could induce apoptosis in HepG2 cells (Figure 3). Cytochrome c has been identified as an important mediator in apoptotic pathways. 24 The release of mitochondrial cytochrome c into the cytoplasm stimulates apoptosis and is commonly used as an indicator of the apoptotic process in the cell.25 In order to confirm the apoptosis activity of these trichothecenes, cytochrome c releasing assays were carried out. The results confirmed that 2′,3′-epoxymyrothecine A, 1, myrothecine A, 3, 14′-hydroxymytoxin B, 5, vertisporin, 6, and roridin E, 8, could promote the cytochrome c release from the mitochondria and subsequently cause apoptosis in HepG2 cells (Figure 4). Cell cycle regulation in the G1 phase has attracted a great deal of attention as a promising target for the research and treatment of cancer. Many of the important genes associated with G1 regulation have been shown to play a key role in proliferation, differentiation and oncogenic transformation, and 5997



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Figure 4. Trichothecene-induced release of cytochrome c in the HepG2 cell line. Cells were treated with 5-Fu (50 μM), 1 (50 μM), 3 (50 μM), 5 (50 nM), 6 (50 nM), and 8 (50 nM) for 12 h.

Figure 5. Trichothecene-induced G1 cell cycle arrest in the HepG2 cell line. Cells were treated with 5-Fu (50 μM), compounds 1 (50 μM), 2 (50 nM), 3 (50 μM), 4 (5 nM), 5-8 (50 nM), and 9 (1 μM) for 12 h).

hydroxy group located at C-13′ of 2 became a keto group in 5. These results revealed that the keto group located at C-13′ was an important site for their activity in HepG2 cells (Figure 3).

For trichothecenes 8 and 9, the only difference between them was the hydroxy group at C-12′. Roridin E, 8, exhibited far stronger cytotoxicity than its derivative 12′-hydroxyroridin 5998



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Figure 6. PARP-cleavage and protein JKN phosphorylation in treated MCF-7 cells, A549 cells, and HepG2 cells. Cells were incubated at concentration 50 μM of compound 1 for 0, 2, 4, and 8 h, respectively, or at concentrations 0, 20, 50, and 100 μM of compound 1 for 8 h.

Funding

E, 9, and 12′-hydroxyroridin E, 9, which exhibited no activity on PARP-cleavage and phosphorylation of JNK protein in HepG2 cells (Figure 3). These results indicated that hydroxy group substitution at C-12′ was vital for PARP-cleavage and phosphorylation of JNK protein in HepG2 cells. 2′,3′-Epoxymyrothecine A could induce the PARP-cleavage in a time- and dose-dependent manner in HepG2 and MCF-7 cells (Figure 6). Compared to HepG2 cells, 2′,3′-epoxymyrothecine A exhibited clear PARP-cleavage only after 8 h in MCF-7 cells, and the dose-dependency was much weaker (Figure 6). 2′,3′-Epoxymyrothecine A did not exhibit the same time-dependent effect on the PARP-cleavage in A549 cells. When A549 cells were treated with 2′,3′-epoxymyrothecine A (50 μM) for 8 h, the PARP-cleavage was unclear (Figure 6). For the dose-dependent experiment, 2′,3′-epoxymyrothecine A did not showed any PARP-cleavage activity until the concentration increased to 100 μM (Figure 6). All these results revealed that 2′,3′-epoxymyrothecine A showed much better apoptosis activity in HepG2 cells compared to A549 and MCF-7 cells. 2′,3′-Epoxymyrothecine A can induce phosphorylation of JNK protein in a time- and dose-dependent manner in HepG2 and MCF-7 cells (Figure 6). There were no significant differences of phosphorylation of JNK protein when A549 cells were treated with 50 μM 2′,3′-epoxymyrothecine A for 2, 4, and 8 h. In terms of dose-dependent manner, it seems that 50 μM 2′,3′-epoxymyrothecine A would achieve the plateau (Figure 6). From the perspective of JNK phosphorylation, 2′,3′-epoxymyrothecine A was more suitable for the treatment of HepG2 and MCF-7 cells. Based on these results, we can conclude that 2′,3′-epoxymyrothecine A can induce the G1 cell cycle arrest, phosphorylation of JNK protein, and the PARPcleavage in a time- and dose-dependent manner, promote the cytochrome c release from the mitochondria, and eventually induce apoptosis in HepG2 liver cancer cells. All these results indicated that a small change in trichothecenes could dramatically reduce the toxicity and still retain the apoptosis inducing activity in human cancer cells. These data provide a pilot study for future exploitation of the applicability of trichothecenes in cancer chemotherapy.

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This study were supported by the Natural Science Foundation of Fujian Province of China (No. 2011J01251) and National Natural Science Foundation of China (No. 81202439). Notes

The authors declare no competing financial interest.

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(1) He, J. W.; Zhou, T.; Christopher, Y. J.; Boland, G. J.; Scottc, P. M. Chemical and biological transformations for detoxification of trichothecene mycotoxins in human and animal food chains: a review. Trends Food. Sci. Technol. 2010, 21, 67−76. (2) McCormick, S. P; Alexander, N. J; Proctor, R. H. Trichothecene Triangle: Toxins, Genes, and Plant Disease. In Phytochemicals, Plant Growth,and the Environment; Gang, D. R., Bernards, M. A., Davin, L. B., Jetter, R., McCormick, S. P., Stevens, J. F., Eds.; Springer: New York, 2013; Vol. 42, pp 1−2. (3) Ueno, Y. Trichothecene mycotoxins: Mycology, chemistry, and toxicology. Adv. Nutr. Res. 1989, 3, 301−353. (4) Lancova, K.; Hajslova, J.; Poustka, J.; Krplova, A.; Zachariasova, M.; Dostalek, P.; Sachambula, L. Transfer of Fusarium mycotoxins and ‘masked’ deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer. Food Addit. Contam. A 2008, 25, 732− 744. (5) Alvi, K. A.; Rabenstein, J.; Woodard, J.; Baker, D. D.; Bergthold, J. D.; Lynch, J.; Lieu, K. L.; Braude, I. A. 14′-Hydroxymytoxin B and 16hydroxyroridin E, two new cytotoxic trichothecenes from Myrothecium roridum. J. Nat. Prod. 2002, 65, 742−744. (6) Abbas, H. K.; Yoshizawa, T.; Shier, W. T. Cytotoxicity and phytotoxicity of trichothecene mycotoxins produced by Fusarium spp. Toxicon 2013, 74, 68−75. (7) Isaka, M.; Punya, J.; Lertwerawat, Y.; Tanticharoen, M.; Thebtaranonth, Y. Antimalarial activity of macrocyclic trichothecenes isolated from the fungus Myrothecium verrucaria. J. Nat. Prod. 1999, 62, 329−331. (8) Bondy, G. S.; Pestka, J. J. Immunomodulation by fungal toxins. J. Toxicol. Environ. Health, Part B 2000, 3, 109−143. (9) Amagata, T.; Rath, C.; Rigot, J. F.; Tarlov, N.; Tenney, K.; Valeriote, F. A.; Crews, P. Structures and cytotoxic properties of trichoverroids and their macrolide analogues produced by saltwater culture of Myrothecium verrucaria. J. Med. Chem. 2003, 46, 4342−4350. (10) Claridge, C. A.; Schmitz, H.; Bradner, W. T. Antitumor activity of some microbial and chemical transformation products of anguidine (4,15-diacetoxyscirpene-3-ol). Cancer Chemother. Pharmacol. 1979, 2, 181−182. (11) Thompson, W. L.; Wannemacher, R. W., Jr. Detection and quantitation of T-2 mycotoxin with a simplified protein synthesis inhibition assay. Appl. Environ. Microbiol. 1984, 48, 1176−1180. (12) Shen, L.; Jiao, R. H.; Ye, Y. H.; Wang, X. T.; Xu, C.; Song, Y. C.; Zhu, H. L.; Tan, R. X. Absolute configuration of new cytotoxic and other bioactive trichothecene macrolides. Chemistry 2006, 12, 5596− 5602. (13) Jarvis, B. B.; Comezoglu, F. T.; Wang, S.; Ammon, H. L. Myrotoxins from a plant pathogenic isolate of Myrothecium roridum. Mycotoxin Res. 1991, 7, 73−78.

ASSOCIATED CONTENT

* Supporting Information S

1D and 2D NMR and HR-MS of compounds 1 and 2. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

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Corresponding Author

*Phone: 86-592-2187225. Fax: 86-592-2181879. E-mail: [email protected]. 5999



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(14) Hayakawa, S.; Kondo, E.; Wakisaka, Y.; Minato, H.; Katagiri, K. Vertisporin, a new antibiotic from Verticimonosporium dif f ractum. J. Antibiot. (Tokyo) 1975, 28, 550−551. (15) Jarvis, B. B.; Taha Ç ömezoglu, F.; Lee, Y. W.; FlippenAnderson, J. L.; Gilardi, R. D.; George, C. F. Novel Macrocyclic Trichothecenes from Myrothecium Roridum. Bull. Soc. Chim. Belges. 1986, 95, 681−697. (16) Traxler, P.; Zurcher, W.; Tamm, C. Structure of the antibiotic roridin E. Helv. Chim. Acta 1970, 53, 2071−2085. (17) Xu, J.; Takasaki, A.; Kobayashi, H.; Oda, T.; Yamada, J.; Mangindaan, R. E.; Ukai, K.; Nagai, H.; Namikoshi, M. Four new macrocyclic trichothecenes from two strains of marine-derived fungi of the genus Myrothecium. J. Antibiot. (Tokyo) 2006, 59, 451−455. (18) Pestka, J. J.; Zhou, H. R.; Moon, Y.; Chung, Y. J. Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other trichothecenes: unraveling a paradox. Toxicol. Lett. 2004, 153, 61−73. (19) Kobayashi, M.; Kanasaki, R.; Ezaki, M.; Sakamoto, K.; Takase, S.; Fujie, A.; Hino, M.; Hori, Y. FR227244, a novel antifungal antibiotic from Myrothecium cinctum No. 002 Taxonomy, fermentation, isolation and physico-chemical properties. J. Antibiot. (Tokyo) 2004, 57, 780−787. (20) Abbas, H. K.; Johnson, B. B.; Shier, W. T.; Tak, H.; Jarvis, B. B.; Boyette, C. D. Phytotoxicity and mammalian cytotoxicity of macrocyclic trichothecene mycotoxins from Myrothecium verrucaria. Phytochemistry 2002, 59, 309−313. (21) Steffen, J. D.; Tholey, R. M.; Langelier, M. F.; Planck, J. L.; Schiewer, M. J.; Lal, S.; Bildzukewicz, N. A.; Yeo, C. J.; Knudsen, K. E.; Brody, J. R.; Pascal, J. M. Targeting PARP-1 allosteric regulation offers therapeutic potential against cancer. Cancer Res. 2013, 74, 31−37. (22) Nguewa, P. A.; Fuertes, M. A.; Valladares, B.; Alonso, C.; Perez, J. M. Poly(ADP-ribose) polymerases: homology, structural domains and functions. Novel therapeutical applications. Prog. Biophys. Mol. Biol. 2005, 88, 143−172. (23) Germain, M.; Affar, E. B.; D’Amours, D.; Dixit, V. M.; Salvesen, G. S.; Poirier, G. G. Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7. J. Biol. Chem. 1999, 274, 28379−28384. (24) Bouchard, V. J.; Rouleau, M.; Poirier, G. G. PARP-1, a determinant of cell survival in response to DNA damage. Exp. Hematol. 2003, 31, 446−454. (25) Liu, X.; Kim, C. N.; Yang, J.; Jemmerson, R.; Wang, X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 1996, 86, 147−157. (26) Delaval, B.; Birnbaum, D. A cell cycle hypothesis of cooperative oncogenesis (Review). Int. J. Oncol. 2007, 30, 1051−1058. (27) Wada, T.; Penninger, J. M. Mitogen-activated protein kinases in apoptosis regulation. Oncogene 2004, 23, 2838−2849. (28) Tournier, C. The 2 faces of JNK Signaling in cancer. Genes Cancer 2013, 4, 397−400. (29) Dhanasekaran, D. N.; Johnson, G. L. MAPKs: function, regulation, role in cancer and therapeutic targeting. Oncogene 2007, 26, 3097−3099. (30) Dhanasekaran, N.; Premkumar, R. E. Signaling by dual specificity kinases. Oncogene 1998, 17, 1447−1455. (31) Seimiya, H.; Mashima, T.; Toho, M.; Tsuruo, T. c-Jun NH2terminal kinase-mediated activation of interleukin-1beta converting enzyme/CED-3-like protease during anticancer drug-induced apoptosis. J. Biol. Chem. 1997, 272, 4631−4636. (32) Waskiewicz, A. J.; Cooper, J. A. Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast. Curr. Opin. Cell Biol. 1995, 7, 798−805. (33) Tibbles, L. A.; Woodgett, J. R. The stress-activated protein kinase pathways. Cell. Mol. Life Sci. 1999, 55, 1230−1254. (34) Davis, R. J. Signal transduction by the JNK group of MAP kinases. Cell 2000, 103, 239−252. (35) Chang, L.; Karin, M. Mammalian MAP kinase signalling cascades. Nature 2001, 410, 37−40.

(36) Johnson, G. L.; Lapadat, R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002, 298, 1911−1912. (37) Werlen, G.; Hausmann, B.; Naeher, D.; Palmer, E. Signaling life and death in the thymus: timing is everything. Science 2003, 299, 1859−1863. (38) Ventura, J. J.; Hubner, A.; Zhang, C.; Flavell, R. A.; Shokat, K. M.; Davis, R. J. Chemical genetic analysis of the time course of signal transduction by JNK. Mol. Cell 2006, 21, 701−710.

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