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Myrotheciumones: Bicyclic cytotoxic lactones isolated from an endophytic fungus of Ajuga decumbens
Bioorganic & Medicinal Chemistry Letters 24 (2014) 2504–2507
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Myrotheciumones: Bicyclic cytotoxic lactones isolated from an endophytic fungus of Ajuga decumbens Ting Lin ⇑, , Guanghui Wang , Wenjun Shan , Dequan Zeng, Rong Ding, Xin Jiang, Dan Zhu, Xiaoxuan Liu, Shuiyuan Yang, Haifeng Chen ⇑ School of Pharmaceutical Sciences, Xiamen University, South Xiangan Road, Xiamen, Fujian 361102, China
a r t i c l e
i n f o
Article history: Received 11 December 2013 Revised 17 March 2014 Accepted 3 April 2014 Available online 13 April 2014 Keywords: Myrotheciumones Endophytic fungus Ajuga decumbens Cytotoxicity Apoptosis
a b s t r a c t Two new bicyclic lactones, myrotheciumones A (1) and B (2) which possessed a rare ring-fusion system were isolated from Myrothecium roridum (M. roridum), an endophytic fungus of the medicinal herb plant Ajuga decumbens (A. decumbens) via an in vitro cytotoxicity assay. Structures were deduced from 1D and 2D NMR (Nuclear magnetic resonance) data. Myrotheciumone A’s in vitro cytotoxicity and apoptotic activity were evaluated and myrotheciumone A was shown to exert cytotoxicity via inducing apoptosis in cancer cell line. Ó 2014 Elsevier Ltd. All rights reserved.
Cancer, characterized by uncontrolled growth of abnormal cells and invasion into normal tissue, is a group of diverse diseases affecting various organs of the body. Cancers that metastasize are of particular concern because they can produce new tumors at distant sites.1 Current cancer therapies: chemotherapy, c-irradiation, immunotherapy, or suicide gene therapy, primarily exert antitumor effects by triggering cancer cell apoptosis,2,3 and this mechanisms offers promise for future cancer drug discoveries.4 In the past half-century since the ‘Golden Age of Antibiotic Discovery’, natural products have gradually been marginalized by major pharmaceutical research companies as sources for new drugs.5 Natural products, especially from plant-associated microbes, are thus largely unexplored.6,7 Endophytic fungi, which live symbiotically within higher plant tissues,1 occur in all plants studied to date. Of the more than 300,000 higher plant species, all can be assumed to host complex communities of endophytic microbes,8 which are significant reservoirs of genetic diversity and important sources for the discovery of novel bioactive secondary metabolites.9 After an initial report of the production of paclitaxel from an endophyte of the Northwest Pacific yew,10 isolation of anticancer agents from fungal endophytes has gained increased interest. For example, paclitaxel,10 camptothecin,11 vincristine,12 and podophyllotoxin,13 have been ⇑ Corresponding authors. Tel.: +86 592 288 1180; fax: +86 592 218 1879.
E-mail addresses: [email protected] (T. Lin), [email protected] (H. Chen). These three authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.04.016 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
marked, and numerous anticancer compounds with unique structures have also been isolated, chaetopyranin (aldehyde),14 9-deacetoxyfumigaclavine C (alkaloid),15 saldinone C and D (fluoranthenes),16 pestalotiopsone F (chromone),17 beauvericin (depsipeptide),18 ergoflavin (ergochromes),19 globosumone A and B (ester),20 eutypellin A (lactone),21 leucinostatin A (peptide),22 penicillenone (polyketide),23 periconicin B (diterpene),24 tauranin (sesquiterpene),25 and phomoxanthones A and B (xanthone).26 Thus, endophytic fungi have been shown to offer many anticancer compounds of diverse chemical classes.1 Ajuga decumbens (A. decumbens) is a naturally occurring herb in Japan and China.27 The flowering whole plants of A. decumbens have been used in folk medicine for antiinflammatory, antitussive, and expectorant effects.28,29 The main bioactive components of A. decumbens are iridoid glycosides, which have been isolated the treatment of chronic pelvic inflammation and hysteromyoma.30–32 Many bioactive compounds have been isolated and identified from the whole plant,33–38 several of which have anti-tumor activity.39 In the course of secondary metabolite chemotherapeutic screening of endophytic fungi isolated from A. decumbens, we confirmed that activity in Myrothecium roridum (M. roridum) was isolated from the stem of the A. decumbens. M. roridum was identified by ITS (Internal Transcribed Spacer) method. We isolated and characterized the structure of two new bicyclic lactones, and myrotheciumones A (1) was evaluated for in vitro cytotoxicity and apoptosis in the HepG2 (hepatocellular carcinoma) cancer cell line.
T. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2504–2507
2505
Table 1 NMR data of myrotheciumones A (1) and B (2) in CDCl3a No.
1 dC
1 2
dH, mult. (J in Hz)
dC
dH, mult. (J in Hz)
a 2.82, dd (18.5, 4.1)
175.5 58.0
2.92, d (4.3)
b 2.51, dd (18.5, 12.1) 3.09, m 1.89, m
3 4 5
42.1 45.1 79.6
6
47.8
a 2.22, d (15.2)
68.6
a 1.78, dd (14.0, 3.2) b 2.27, dd (14.0, 8.9) 4.65, ddd (8.6, 4.0, 3.4)
83.4 8.7 25.5
b 1.92, dd (14.9, 6.6) 5.07, t (7.1) 1.06, d (7.0) 1.28, s
9.3 16.2
1.04, d (6.8) 1.42, s
7 8 9 a
178.3 30.1
2
48.0 90.3 43.9
1.99, q (6.7)
Data were measured in CDCl3 at 600 MHz (1H) and 100 MHz (13C).
Figure 1. Selected HMBC and NOSEY correlations for 1 and 2.
An ethyl acetate extract of a fermented product of M. roridum was separated by chromatography over silica gel and RP-18 (reverse phase C-18) column, yielding two new bicyclic lactones, myrotheciumones A (1) and B (2). Myrotheciumone A (1), isolated as colorless oil had the following characteristics according to IR (KBr) (mmax): 3452 (OAH), 1754 (C@O). The molecular formula: C9H14O3 according to HR-FT-MS
Figure 3. Myrotheciumone A (1)-induced activity of caspase-3 in HepG2 cells (treated with compound 1, 4 lM, for 0, 3, 6, and 12 h, respectively).
data (m/z 193.0838 [M+Na]+). 1H and 13C data see Table 1. 1H NMR data and the HSQC spectrum confirmed the presence of two methyl groups (a singlet at d 1.28 and a doublet at d 1.06), two methylenes, three methines, one oxygenated quaternary carbon group and one carbonyl group. 1 H–1H COSY correlations between H-3 and H-2, H-3 and H-4, H-3 and H-7, and H-7 and H-6 established the structure of a 5-carbon moiety. Key HMBC correlations from methyl protons at d 1.06 to three carbons at d 42.1 (C-3), 45.1 (C-4) and 79.6 (C-5), from methyl protons at d 1.29 to three carbons at d 45.1 (C-4), 79.6 (C-5) and 47.9 (C-6), from protons at d 3.09 and 5.07 to carbon at d 178.3 (C-1) established the planar structure of 1. The relative configuration of 1 was determined by NOESY spectroscopic data (Fig. 1). The presence of NOEs between H-2a and H-8 indicated the a-orientations of these protons. Furthermore, NOESY correlations between H-2b and H-3, H-3 and H-7, H-3 and H-4, H-4 and H-9, and H-6 and H-7 established the b-orientations of these protons. Therefore, the structure of myrotheciumone A (1) was confirmed. Myrotheciumone B (2), isolated as colorless oil had the following characteristics according to IR (KBr) (mmax): 3416 (OAH), 1765 (C@O). The molecular formula C8H12O3 was deduced from HR-FTMS and 13C NMR. 1H and 13C data see Table 1. Myrotheciumone B was smaller than 1 by a CH2 unit. 1H NMR data for 2 was similar with that of 1 except for the absence of the methylene at d 2.82, 2.51. Interpretation of HSQC, HMBC and COSY data of 2 suggested that the c lactone in 1 was a b lactone in 2. The relative configuration of 2 was determined by NOESY spectroscopic data (Fig. 1). NOESY correlations between H-2 and H-6, H-3 and H-8, H-8 and
Figure 2. PARP cleavage in treated HepG2 cells and statistical results of them. Cells were incubated with compound 1 (5, 10, and 20 lM) for 8 h, or 4 lM of compound 1 for 0, 3, 6, or 12 h, respectively.
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T. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2504–2507
Figure 4. Myrotheciumone A (1)-induced releasing of cytochrome c in HepG2 cells (treated with compound 1, 4 lM, for 12 h).
H-5b, H-5b and H-6, and H-6 and H-3 established the b-orientations of these protons. Therefore, the structure of myrotheciumone B (2) was confirmed. The cytotoxic activities of both compounds in HepG2 (hepatocellular carcinoma, ATCC), SMMC-7721 (human hepatocellular carcinoma, Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China), A549 (human lung adenocarcinoma, ATCC), MCF-7 (human breast adenocarcinoma, ATCC) lines were tested, QSG-7701 (human hepatocyte cell, Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) and HL-7702 (human hepatocyte cell, Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) cell lines were tested40 and data confirmed that myrotheciumone A (1) strongly inhibited growth of HepG2 (IC50: 5.36 ± 0.26 lM), SMMC-7721 (IC50: 6.56 ± 0.58 lM), A549 (IC50: 5.88 ± 0.68 lM), MCF-7 cells (IC50: 7.56 ± 0.76 lM), QSG-7701 (IC50: 16.30 ± 0.31 lM) and HL-7702 (IC50: 20.69 ± 4.69 lM). These data indicated that myrotheciumone A (1) possess more cell-specific cytotoxicity to target on cancer cells rather than normal cells. Myrotheciumone B (2) had no activity against any studied cancer cell lines at 100 lM. Poly (ADP-ribose) polymerase (PARP) has been shown to undergo proteolytic cleavage into 89- and 24-kDa fragments that contain the active site and the DNA-binding domain of the enzyme, respectively, during drug-induced41 and spontaneous apoptosis.42,43 Cleavage of PARP-1 by caspases is a hallmark of apoptosis.41,44 We examined the apoptotic effect of myrotheciumone A (1) on PARP cleavage at the protein level (HepG2). Data showed that myrotheciumone A (1) induced PARP cleavage in a timeand dose-dependent manner (Fig. 2). Then, we also tested the caspase-3 activity in order to double confirm the induction of caspase-mediated PARP cleavage. Caspase-3 activities were determined by a colorimetric assay based on the ability of caspase-3 to change acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) into a yellow formazan product [p-nitroaniline (pNA)]. After treatment with myrotheciumone A (4 lM) for different period (0, 3, 6, and 12 h), assays were performed follow the manufacturer’s protocol (Biovision Research Products). As the time passed, the caspase-3 activation was synergistically induced to reach almost sixfold at 12 h (Fig. 3). Release of key mitochondrial proteins such as cytochrome c is an important hallmark in the pathway of
apoptosis and is considered a point of no return in the apoptotic process.45–48 We observed myrotheciumone A (1)-induced released of cytochrome c in HepG2 cells suggesting that myrotheciumone A (1) dramatically induced cytochrome c release in HepG2 cells (Fig. 4). Thus, we conclude that myrotheciumone A (1) can induce apoptosis in HepG2 cells. In summary, two new bicyclic lactones, myrotheciumones A (1) and B (2) were isolated from the secondary metabolites of M. roridum, an endophytic fungus from A. decumbens. This is the first time the cis-fused 4–5 ring system has been described in natural products. Myrotheciumone A (1) possesses anticancer activity (IC50 5.36–7.56 lM) in four different cancer cell lines and induces the PARP cleavage in a time and dose-dependent manner. Myrotheciumone A (1) showed more cell-specific cytotoxicity to target on cancer cells rather than normal cells. Moreover, myrotheciumone A (1) promotes cytochrome c release from mitochondria. Data show that myrotheciumone A (1) had cytotoxicity by inducing apoptosis in cancer cell lines. These data provide a strong foundation for future studies to exploit the promise of myrotheciumone A (1) for cancer chemotherapy. Acknowledgments This work were supported by the Xiamen Science and Technology Key program grant, China (No. 3502Z20100006), Natural Science Foundation of Fujian Province of China (No. 2011J01251) and National Natural Science Foundation of China (No. 81202439). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 04.016. References and notes 1. Kharwar, R. N.; Mishra, A.; Gond, S. K.; Stierle, A.; Stierle, D. Nat. Prod. Rep. 2011, 28, 1208. 2. Makin, G.; Dive, C. Trends Cell Biol. 2001, 11, 22. 3. Fulda, S.; Debatin, K. M. Curr. Cancer Drug Targets 2004, 4, 569. 4. Fesik, S. W. Nat. Rev. Cancer 2005, 5, 876.
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Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Myrotheciumones: Bicyclic cytotoxic lactones isolated from an endophytic fungus of Ajuga decumbens Ting Lin ⇑, , Guanghui Wang , Wenjun Shan , Dequan Zeng, Rong Ding, Xin Jiang, Dan Zhu, Xiaoxuan Liu, Shuiyuan Yang, Haifeng Chen ⇑ School of Pharmaceutical Sciences, Xiamen University, South Xiangan Road, Xiamen, Fujian 361102, China
a r t i c l e
i n f o
Article history: Received 11 December 2013 Revised 17 March 2014 Accepted 3 April 2014 Available online 13 April 2014 Keywords: Myrotheciumones Endophytic fungus Ajuga decumbens Cytotoxicity Apoptosis
a b s t r a c t Two new bicyclic lactones, myrotheciumones A (1) and B (2) which possessed a rare ring-fusion system were isolated from Myrothecium roridum (M. roridum), an endophytic fungus of the medicinal herb plant Ajuga decumbens (A. decumbens) via an in vitro cytotoxicity assay. Structures were deduced from 1D and 2D NMR (Nuclear magnetic resonance) data. Myrotheciumone A’s in vitro cytotoxicity and apoptotic activity were evaluated and myrotheciumone A was shown to exert cytotoxicity via inducing apoptosis in cancer cell line. Ó 2014 Elsevier Ltd. All rights reserved.
Cancer, characterized by uncontrolled growth of abnormal cells and invasion into normal tissue, is a group of diverse diseases affecting various organs of the body. Cancers that metastasize are of particular concern because they can produce new tumors at distant sites.1 Current cancer therapies: chemotherapy, c-irradiation, immunotherapy, or suicide gene therapy, primarily exert antitumor effects by triggering cancer cell apoptosis,2,3 and this mechanisms offers promise for future cancer drug discoveries.4 In the past half-century since the ‘Golden Age of Antibiotic Discovery’, natural products have gradually been marginalized by major pharmaceutical research companies as sources for new drugs.5 Natural products, especially from plant-associated microbes, are thus largely unexplored.6,7 Endophytic fungi, which live symbiotically within higher plant tissues,1 occur in all plants studied to date. Of the more than 300,000 higher plant species, all can be assumed to host complex communities of endophytic microbes,8 which are significant reservoirs of genetic diversity and important sources for the discovery of novel bioactive secondary metabolites.9 After an initial report of the production of paclitaxel from an endophyte of the Northwest Pacific yew,10 isolation of anticancer agents from fungal endophytes has gained increased interest. For example, paclitaxel,10 camptothecin,11 vincristine,12 and podophyllotoxin,13 have been ⇑ Corresponding authors. Tel.: +86 592 288 1180; fax: +86 592 218 1879.
E-mail addresses: [email protected] (T. Lin), [email protected] (H. Chen). These three authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.04.016 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
marked, and numerous anticancer compounds with unique structures have also been isolated, chaetopyranin (aldehyde),14 9-deacetoxyfumigaclavine C (alkaloid),15 saldinone C and D (fluoranthenes),16 pestalotiopsone F (chromone),17 beauvericin (depsipeptide),18 ergoflavin (ergochromes),19 globosumone A and B (ester),20 eutypellin A (lactone),21 leucinostatin A (peptide),22 penicillenone (polyketide),23 periconicin B (diterpene),24 tauranin (sesquiterpene),25 and phomoxanthones A and B (xanthone).26 Thus, endophytic fungi have been shown to offer many anticancer compounds of diverse chemical classes.1 Ajuga decumbens (A. decumbens) is a naturally occurring herb in Japan and China.27 The flowering whole plants of A. decumbens have been used in folk medicine for antiinflammatory, antitussive, and expectorant effects.28,29 The main bioactive components of A. decumbens are iridoid glycosides, which have been isolated the treatment of chronic pelvic inflammation and hysteromyoma.30–32 Many bioactive compounds have been isolated and identified from the whole plant,33–38 several of which have anti-tumor activity.39 In the course of secondary metabolite chemotherapeutic screening of endophytic fungi isolated from A. decumbens, we confirmed that activity in Myrothecium roridum (M. roridum) was isolated from the stem of the A. decumbens. M. roridum was identified by ITS (Internal Transcribed Spacer) method. We isolated and characterized the structure of two new bicyclic lactones, and myrotheciumones A (1) was evaluated for in vitro cytotoxicity and apoptosis in the HepG2 (hepatocellular carcinoma) cancer cell line.
T. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2504–2507
2505
Table 1 NMR data of myrotheciumones A (1) and B (2) in CDCl3a No.
1 dC
1 2
dH, mult. (J in Hz)
dC
dH, mult. (J in Hz)
a 2.82, dd (18.5, 4.1)
175.5 58.0
2.92, d (4.3)
b 2.51, dd (18.5, 12.1) 3.09, m 1.89, m
3 4 5
42.1 45.1 79.6
6
47.8
a 2.22, d (15.2)
68.6
a 1.78, dd (14.0, 3.2) b 2.27, dd (14.0, 8.9) 4.65, ddd (8.6, 4.0, 3.4)
83.4 8.7 25.5
b 1.92, dd (14.9, 6.6) 5.07, t (7.1) 1.06, d (7.0) 1.28, s
9.3 16.2
1.04, d (6.8) 1.42, s
7 8 9 a
178.3 30.1
2
48.0 90.3 43.9
1.99, q (6.7)
Data were measured in CDCl3 at 600 MHz (1H) and 100 MHz (13C).
Figure 1. Selected HMBC and NOSEY correlations for 1 and 2.
An ethyl acetate extract of a fermented product of M. roridum was separated by chromatography over silica gel and RP-18 (reverse phase C-18) column, yielding two new bicyclic lactones, myrotheciumones A (1) and B (2). Myrotheciumone A (1), isolated as colorless oil had the following characteristics according to IR (KBr) (mmax): 3452 (OAH), 1754 (C@O). The molecular formula: C9H14O3 according to HR-FT-MS
Figure 3. Myrotheciumone A (1)-induced activity of caspase-3 in HepG2 cells (treated with compound 1, 4 lM, for 0, 3, 6, and 12 h, respectively).
data (m/z 193.0838 [M+Na]+). 1H and 13C data see Table 1. 1H NMR data and the HSQC spectrum confirmed the presence of two methyl groups (a singlet at d 1.28 and a doublet at d 1.06), two methylenes, three methines, one oxygenated quaternary carbon group and one carbonyl group. 1 H–1H COSY correlations between H-3 and H-2, H-3 and H-4, H-3 and H-7, and H-7 and H-6 established the structure of a 5-carbon moiety. Key HMBC correlations from methyl protons at d 1.06 to three carbons at d 42.1 (C-3), 45.1 (C-4) and 79.6 (C-5), from methyl protons at d 1.29 to three carbons at d 45.1 (C-4), 79.6 (C-5) and 47.9 (C-6), from protons at d 3.09 and 5.07 to carbon at d 178.3 (C-1) established the planar structure of 1. The relative configuration of 1 was determined by NOESY spectroscopic data (Fig. 1). The presence of NOEs between H-2a and H-8 indicated the a-orientations of these protons. Furthermore, NOESY correlations between H-2b and H-3, H-3 and H-7, H-3 and H-4, H-4 and H-9, and H-6 and H-7 established the b-orientations of these protons. Therefore, the structure of myrotheciumone A (1) was confirmed. Myrotheciumone B (2), isolated as colorless oil had the following characteristics according to IR (KBr) (mmax): 3416 (OAH), 1765 (C@O). The molecular formula C8H12O3 was deduced from HR-FTMS and 13C NMR. 1H and 13C data see Table 1. Myrotheciumone B was smaller than 1 by a CH2 unit. 1H NMR data for 2 was similar with that of 1 except for the absence of the methylene at d 2.82, 2.51. Interpretation of HSQC, HMBC and COSY data of 2 suggested that the c lactone in 1 was a b lactone in 2. The relative configuration of 2 was determined by NOESY spectroscopic data (Fig. 1). NOESY correlations between H-2 and H-6, H-3 and H-8, H-8 and
Figure 2. PARP cleavage in treated HepG2 cells and statistical results of them. Cells were incubated with compound 1 (5, 10, and 20 lM) for 8 h, or 4 lM of compound 1 for 0, 3, 6, or 12 h, respectively.
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Figure 4. Myrotheciumone A (1)-induced releasing of cytochrome c in HepG2 cells (treated with compound 1, 4 lM, for 12 h).
H-5b, H-5b and H-6, and H-6 and H-3 established the b-orientations of these protons. Therefore, the structure of myrotheciumone B (2) was confirmed. The cytotoxic activities of both compounds in HepG2 (hepatocellular carcinoma, ATCC), SMMC-7721 (human hepatocellular carcinoma, Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China), A549 (human lung adenocarcinoma, ATCC), MCF-7 (human breast adenocarcinoma, ATCC) lines were tested, QSG-7701 (human hepatocyte cell, Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) and HL-7702 (human hepatocyte cell, Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) cell lines were tested40 and data confirmed that myrotheciumone A (1) strongly inhibited growth of HepG2 (IC50: 5.36 ± 0.26 lM), SMMC-7721 (IC50: 6.56 ± 0.58 lM), A549 (IC50: 5.88 ± 0.68 lM), MCF-7 cells (IC50: 7.56 ± 0.76 lM), QSG-7701 (IC50: 16.30 ± 0.31 lM) and HL-7702 (IC50: 20.69 ± 4.69 lM). These data indicated that myrotheciumone A (1) possess more cell-specific cytotoxicity to target on cancer cells rather than normal cells. Myrotheciumone B (2) had no activity against any studied cancer cell lines at 100 lM. Poly (ADP-ribose) polymerase (PARP) has been shown to undergo proteolytic cleavage into 89- and 24-kDa fragments that contain the active site and the DNA-binding domain of the enzyme, respectively, during drug-induced41 and spontaneous apoptosis.42,43 Cleavage of PARP-1 by caspases is a hallmark of apoptosis.41,44 We examined the apoptotic effect of myrotheciumone A (1) on PARP cleavage at the protein level (HepG2). Data showed that myrotheciumone A (1) induced PARP cleavage in a timeand dose-dependent manner (Fig. 2). Then, we also tested the caspase-3 activity in order to double confirm the induction of caspase-mediated PARP cleavage. Caspase-3 activities were determined by a colorimetric assay based on the ability of caspase-3 to change acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) into a yellow formazan product [p-nitroaniline (pNA)]. After treatment with myrotheciumone A (4 lM) for different period (0, 3, 6, and 12 h), assays were performed follow the manufacturer’s protocol (Biovision Research Products). As the time passed, the caspase-3 activation was synergistically induced to reach almost sixfold at 12 h (Fig. 3). Release of key mitochondrial proteins such as cytochrome c is an important hallmark in the pathway of
apoptosis and is considered a point of no return in the apoptotic process.45–48 We observed myrotheciumone A (1)-induced released of cytochrome c in HepG2 cells suggesting that myrotheciumone A (1) dramatically induced cytochrome c release in HepG2 cells (Fig. 4). Thus, we conclude that myrotheciumone A (1) can induce apoptosis in HepG2 cells. In summary, two new bicyclic lactones, myrotheciumones A (1) and B (2) were isolated from the secondary metabolites of M. roridum, an endophytic fungus from A. decumbens. This is the first time the cis-fused 4–5 ring system has been described in natural products. Myrotheciumone A (1) possesses anticancer activity (IC50 5.36–7.56 lM) in four different cancer cell lines and induces the PARP cleavage in a time and dose-dependent manner. Myrotheciumone A (1) showed more cell-specific cytotoxicity to target on cancer cells rather than normal cells. Moreover, myrotheciumone A (1) promotes cytochrome c release from mitochondria. Data show that myrotheciumone A (1) had cytotoxicity by inducing apoptosis in cancer cell lines. These data provide a strong foundation for future studies to exploit the promise of myrotheciumone A (1) for cancer chemotherapy. Acknowledgments This work were supported by the Xiamen Science and Technology Key program grant, China (No. 3502Z20100006), Natural Science Foundation of Fujian Province of China (No. 2011J01251) and National Natural Science Foundation of China (No. 81202439). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 04.016. References and notes 1. Kharwar, R. N.; Mishra, A.; Gond, S. K.; Stierle, A.; Stierle, D. Nat. Prod. Rep. 2011, 28, 1208. 2. Makin, G.; Dive, C. Trends Cell Biol. 2001, 11, 22. 3. Fulda, S.; Debatin, K. M. Curr. Cancer Drug Targets 2004, 4, 569. 4. Fesik, S. W. Nat. Rev. Cancer 2005, 5, 876.
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