Search

Activated macrophages contribute prominently to the progression and maintenance of almost all inflammatory and autoimmune diseases. Although non-specific elimination of these phagocytes has been shown to treat animal models of inflammatory disease, the same therapies have been compromised by unacceptable toxicities, because they also kill quiescent macrophages in healthy tissues. In the studies below, we exploit upregulation of folate receptor beta (FRβ) on inflammatory (but not resting) macrophages to target a cytotoxic drug selectively to the inflammatory subset of macrophages. Because many of these activated macrophages are nondividing, we also employ verrucarin A as the cytotoxic payload, since it kills both mitotic and nonmitotic cells by blocking protein synthesis. By inserting a redox-sensitive self-immolative linker between the folate and verrucarin A, we further assure that release of unmodified verrucarin A is triggered primarily after internalization by an FRβ-positive cell. The resulting folate-verrucarin A conjugate is shown to kill FR-expressing cells in vitro in a manner that can be inhibited by competition with 100-fold excess folic acid. The folate-verrucarin A conjugate is also shown to successfully treat a murine model of inflammatory peritonitis by eliminating inflammatory macrophages without killing other cells in the same peritonitis fluid. Based on this high specificity for inflammatory macrophages, we conclude that folate-verrucarin A warrants continued exploration as a potential therapy for inflammatory and autoimmune diseases in humans.

Neuroendocrine (NE) cancers arise from cells within the neuroendocrine system. Chemotherapies and endoradiotherapy have been developed, but their clinical efficacy is limited. The objective of this study was to develop a dual-targeted extracellular vesicles (EV)-delivered combined therapies to treat NE cancer. Specifically, we produced EV in stirred-tank bioreactors and surface tagged both anti-somatostatin receptor 2 (SSTR 2) monoclonal antibody (mAb) and anti-C-X-C motif chemokine receptor 4 (CXCR4) mAb to generate mAbs-EV. Both live-cell confocal microscopy imaging and In Vivo Imaging System (IVIS) imaging confirmed that mAbs-EV specifically targeted and accumulated in NE cancer cells and NE tumor xenografts. Then the highly potent natural cytotoxic marine compound verrucarin A (Ver-A) with IC50 of 2.2&ndash, 2.8 nM and microtubule polymerization inhibitor mertansine (DM1) with IC50 of 3.1&ndash, 4.2 nM were packed into mAbs-EV. The in vivo maximum tolerated dose study performed in non-tumor-bearing mice indicated minimal systemic toxicity of mAbs-EV-Ver-A/DM1. Finally, the in vivo anticancer efficacy study demonstrated that the SSTR2/CXCR4 dual-targeted EV-Ver-A/DM1 is more effective to inhibit NE tumor growth than the single targeting and single drug. The results from this study could expand the application of EV to targeting deliver the combined potent chemotherapies for cancer treatment.

The widespread use of synthetic nematicides has caused significant problems to the environment as well as human health. To address this issue, eco-friendly control measures, such as microbial nematicides, are being developed. During the screening of Myrothecium strains with nematicidal activity against the root-knot nematode (RKN) Meloidogyne incognita, we found that the acetone extract of Myrothecium sp. KACC 40321 was highly effective against hatched juveniles of M. incognita at 7 days after exposure. The fungus was identified as Meloidogyne verrucaria. Two macrocyclic trichothecenes verrucarin A and roridin A were isolated and identified as major active metabolites by bioassay-guided fractionation and instrumental analysis. When the second-stage juveniles were treated with the chemicals, no juvenile mortality was observed. However, they effectively killed juveniles from treated eggs. The hatched juvenile mortality was used to evaluate the in vitro nematicidal activity of the compounds against M. incognita. The median effective concentrations were 1.88 μg/mL for verrucarin A and 1.50 μg/mL for roridin A. Among various liquid media, commercial malt extract broth (cMEB) was found to be the best for the production of verrucarin A and roridin A, followed by potato dextrose broth. The cMEB culture filtrate effectively reduced the formation of galls and egg masses on tomato roots in a pot experiment. In addition, the culture filtrate reduced the formation of galls on the roots of melon plants and the number of RKNs in the soils under field conditions. These results suggest that M. verrucaria KACC 40321 can be used as a biocontrol agent against RKNs in various crops. To the best of our knowledge, this is the first study to report the effectiveness of verrucarin A and rorridin A against hatched juveniles of M. incognita.

The behaviour of Myrothecium roridum, artificially inoculated on cultivated rocket (Eruca sativa), has been evaluated under eight different temperature and CO2 concentration combinations (from 14-18 °C to 26-30 °C and with 400-450 or 800-850 ppm of CO2). The pathogen isolate used for this study was inoculated on rocket and disease severity increased with high temperatures for both CO2 levels. Verrucarin A and roridin E mycotoxins were produced under all the tested temperatures at high CO2 conditions. The maximum level of verrucarin A was found at 14-18 °C and 800-850 ppm of CO2, and the maximum roridin E production was detected at 26-30 °C with 800-850 ppm of CO2. The results obtained in this study show that both the CO2 concentration and the temperature influence disease severity and mycotoxin production in different ways. An increase in temperature, which is favourable for attacks of the pathogen, could induce the spread of M. roridum in temperate regions, and this pathogen could take on even greater importance in the future, considering its ability to produce mycotoxins.

An improved disc diffusion type bioassay was developed for T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), deoxynivalenol (DON), nivalenol (NIV), neosolaniol (NSL), fusarenon-X (FUS-X), trichothecin (TIN), roridin A (RDA) and verrucarin A (VCA) using the yeast, Kluyveromyces marxianus . Factors such as type of medium, agar volume per plate, preincubation time and temperature, incubation temperature, inoculum size and pH had variable, and in some cases a dramatic effect on the sensitivity of the assay. The effect of pH of the assay medium was particularly pronounced. The highest sensitivity was obtained when 6 ml per plate of a tryptic-soy-agar (TSA) medium (pH 7.5) containing 105 CFU inoculum per ml was incubated at 38°C for 18 h. All of the trichothecenes (TNN) were able to inhibit the growth of the yeast with the detection limit being 0.005, 0.01, 0.02, 0.02, 0.1, 0.5, 10, 10, 10 and 50 μg/disc for VCA, RDA, T-2, TTN, DAS, HT-2, DON, NSL, FUS-X and NIV, respectively. The detection limits for corn and wheat spiked with T-2 were 0.1 and 0.2 μg/g, respectively. Non-TNN mycotoxins that did not inhibit yeast at a concentration of 200 μg/disc were aflatoxin B1 (AFB1), ochratoxin A (OA), citrinin (CT), penicillic acid (PA), cyclopiazonic acid (CPA), penitrem-A (PTA) and zearalenone (ZEE). These results indicate that disc diffusion assay using K. marxianus under optimized conditions provides a sensitive method for the detection of low concentrations of several TNN.

The silkworm infection assay is a useful method for directly evaluating the in vivo therapeutic effects of drug candidates. In the present study, 3 known trichothecenes, trichodermin, epiisororidin E, and verrucarin A, were evaluated as antifungal agents in the silkworm-Candida albicans assay. Trichodermin and epiisororidin E yielded effective therapeutic effects, while verrucarin A exhibited no efficacy in this assay system. These results strongly suggest that trichodermin and epiisororidin E are the lead compounds for developing a new antifungal agent.

Endoplasmic reticulum (ER) stress is linked with development and maintenance of cancer, and serves as a therapeutic target for treatment of cancer. Verrucarin A, isolated from the broth of Fusarium sp. F060190, showed potential inhibitory activity on tunicamycin-induced ER stress in FaO rat liver cells. In addition, the compound decreased tunicamycin-induced GRP78 promoter activity in a dose dependent manner without inducing significant inhibition of luciferase activity and cell growth for 6 and 12 h. Moreover, the compound decreased the expression of GRP78, CHOP, XBP-1, and suppressed XBP-1, and reduced phosphorylation of IRE1α in FaO rat liver cells. This evidence suggests for the first time that verrucarin A inhibited tunicamycin-induced ER stress in FaO rat liver cells.

Paradoxically, trichothecenes have both immunosuppressive and immunostimulatory effects. The underlying mechanisms have not been fully explored. Early studies show that dose, exposure timing, and the time at which immune function is assessed influence whether trichothecenes act in an immunosuppressive or immunostimulatory fashion. Recent studies suggest that the immunomodulatory function of trichothecenes is also actively shaped by competing cell-survival and death-signaling pathways. Autophagy may also promote trichothecene immunosuppression, although the mechanism may be complicated. Moreover, trichothecenes may generate an “immune evasion” milieu that allows pathogens to escape host and vaccine immune defenses. Some trichothecenes, especially macrocyclic trichothecenes, also potently kill cancer cells. T-2 toxin conjugated with anti-cancer monoclonal antibodies significantly suppresses the growth of thymoma EL-4 cells and colon cancer cells. The type B trichothecene diacetoxyscirpenol specifically inhibits the tumor-promoting factor HIF-1 in cancer cells under hypoxic conditions. Trichothecin markedly inhibits the growth of multiple cancer cells with constitutively activated NF-κB. The type D macrocyclic toxin Verrucarin A is also a promising therapeutic candidate for leukemia, breast cancer, prostate cancer, and pancreatic cancer. The anti-cancer activities of trichothecenes have not been comprehensively summarized. Here, we first summarize the data on the immunomodulatory effects of trichothecenes and discuss recent studies that shed light on the underlying cellular and molecular mechanisms. These mechanisms include autophagy and major signaling pathways and their crosstalk. Second, the anti-cancer potential of trichothecenes and the underlying mechanisms will be discussed. We hope that this review will show how trichothecene bioactivities can be exploited to generate therapies against pathogens and cancer.

Verrucarin A (VA), an active constituent of pathogenic fungus Myrothecium verrucaria, which has the ability to inhibit the growth of breast cancer cells. However, the mechanism by which VA exerts its inhibitory potential remains elusive. Here, we demonstrated that VA inhibited the growth of MCF-7 breast cancer cells, increased the levels of reactive oxygen species (ROS), and subsequently induced mitochondrial membrane potential (Δψm) loss, leading to the increase of Bax/Bcl-2 ratio, cytochrome c release, caspase activation, PARP degradation, and apoptosis. VA effectively increased the phosphorylation of p38MAPK and diminished the phosphorylation of ERK/Akt. In addition, VA caused cell cycle deregulation through the induction of p21 and p53. Furthermore, ROS scavenger (n-acetyl-L-cysteine) and p38MAPK inhibitor (SB202190) effectively abrogated the VA-induced cell cycle deregulation and apoptosis. Conversely, U0126, an ERK1/2 inhibitor, enhanced the VA-induced apoptotic signals. Taken together, our results suggest that VA-induces apoptosis and cell cycle deregulation in MCF-7 cells through ROS-dependent p38MAPK activation.

The behavior of Myrothecium verrucaria, artificially inoculated on spinach, was studied under seven different temperature conditions (from 5 to 35 °C) and under eight different combinations of temperature and CO2 concentration (14–30 °C and 775–870 or 1550–1650 mg/m3). The isolate used for this study was growing well on spinach, and the mycotoxins verrucarin A and roridin E were produced under all tested temperature and CO2 conditions. The maximum levels of verrucarin A (18.59 ng/g) and roridin E (49.62 ng/g) were found at a temperature of 26–30 °C and a CO2 level of 1550–1650 mg/m3. Rises in temperature as well as in temperature and CO2 concentrations had a significant effect by increasing Myrothecium leaf spots on spinach. The biosynthesis of verrucarin A was significantly increased at the highest temperature (35 °C), while roridin E was influenced by the CO2 concentration. These results show that a positive correlation between climate condition and macrocyclic trichothecene production is possible. However, because of the ability of M. verrucaria to produce mycotoxins, an increase in temperature could induce the spread of M. verrucaria in temperate regions; this pathogen may gain importance in the future.

Verrucarin A (VA) is a small molecule derived from the fungal plant pathogen Myrothecium verrucaria and was identified as a selective inhibitor of clear cell renal cell carcinoma (CCRCC) cell proliferation in a high-throughput screen of a library of naturally occurring small molecules. CCRCC arises as a result of loss-of-function mutations in the von Hippel-Lindau (VHL) gene. Here we show that VA inhibits protein translation initiation culminating in apoptosis through the extrinsic signaling pathway. Reintroduction of the VHL gene in CCRCC cells afforded resistance to VA's apoptotic effects. This resistance is mediated in part by the formation of stress granules that entrap signaling molecules that initiate the apoptotic signaling cascade. The VHL gene product was found to be a component of stress granules that develop as result of VA treatment. These findings reveal an important role for the VHL gene product in cytotoxic stress response and have important implications for the rational development of VA-related compounds in chemotherapeutic targeting of CCRCC.

Two new trichothecenes, named 8α-hydroxyroridin H and myrothecin A, along with six known compounds, 8α-acetoxy roridin H, isororidin K, verrucarin A, verrucarin J, verrucarin L and 8α-acetoxy verrucarin L, were isolated from the fermentation broth of a halotolerant fungus Myrothecium sp. GS-17, which was separated from the soil sample of a salina. Structure elucidation and NMR signal assignments were achieved on the basis of spectroscopy. In addition, compounds 1 and 2 were active against plant pathogenic fungi Rhizoctonia solani and Fusarium oxysporum. Copyright © 2012 John Wiley & Sons, Ltd.

Verrucarin A2 (VerA) is a toxic trichothecene mycotoxin that can be produced indoors at very low level by moulds contaminating dwellings and may be associated with several human health problems. In this study we describe a spectroscopic label-free biosensor for VerA. This sensor is based on the high sensitivity of Fourier transform infrared-attenuated reflection (FTIR-ATR) spectroscopic detection and the use of a new anti-VerA rat monoclonal antibody (mAb). This antibody was directly grafted at the surface of the infrared element. Competitive ELISA and FTIR-ATR techniques were compared for detection of VerA in buffer and in complex dust samples obtained from dwellings. After optimization, the competitive ELISA showed a sensitivity of 7.43 ng/ml of VerA in PBS and a dynamic range below one order of magnitude. The FTIR technique improved the detection of the VerA by three orders of magnitude (2 pg/ml in buffer and 6 pg/ml when spiked in dust samples). The dynamic range for its detection extended over four orders of magnitude. The percentage of recovery of VerA spiked (1000 ng to 0.1 ng) in a complex dust matrix ranged from 99 to 68%. Our results clearly show that this antibody-based spectroscopic biosensor allow a better detection of VerA as compared to classical immunoassays and can be very efficiently used in the field of indoor mycotoxin detection.

Myrothecium verrucaria is a pathogen of several invasive weed species, including kudzu, and is currently being evaluated for use as a bioherbicide. However, the fungus also produces macrocyclic trichothecene mycotoxins. The safety of this biological control agent during production and handling would be improved if an inoculum could be produced without concomitant accumulation of macrocyclic trichothecenes. Sporulation and trichothecene production by M. verrucaria was evaluated on standard potato dextrose agar (PDA) and a series of complex and defined media. Sporulation on PDA and on agar media with nitrogen as ammonium nitrate or potassium nitrate was more than ten-fold greater then sporulation on the medium with ammonium sulphate as the nitrogen source. Accumulation of macrocyclic trichothecenes was strongly affected by the media composition, with higher levels often associated with higher carbon content in the media. Overall, incubation in continuous darkness resulted in higher macrocyclic trichothecene concentrations. Results support the hypothesis that accumulation of macrocyclic trichothecenes by this fungus can be altered by manipulating carbon and nitrogen sources. Furthermore, the biosynthesis of these mycotoxins may be independent of sporulation, demonstrating that the bioherbicide can be readily produced on solid substrates while simultaneously yielding conidia that are less threatening to worker safety. A more detailed implementation of the concepts demonstrated in this study will facilitate the safe and economical production of this bioherbicide.

A rapid technique for trichothecene detection was needed in screening tests of the potential bioherbicide Myrothecium verrucaria (MV), in order to select strains, mutants, or formulations that were void of or that possessed low amounts of these undesirable mycotoxin compounds. Commercially available enzyme‐linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross‐reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and J, roridin A, L‐2, E, and H), were tested for their ability to detect trichothecenes produced by a strain of Myrothecium verrucaria (MV) in cell cultures, in plant tissues (hemp sesbania and kudzu) treated with purified roridin A, or ethyl acetate fractions of MV cultures. Evaluations of ELISA assays showed linear responses for standards of verrucarin A and roridin A over a concentration range of 0.2 to 20 ppb. Ethyl acetate or aqueous extractions were used to obtain samples from MV cultures and plant tissues for testing. Trichothecenes were de...

A mycelial formulation of the fungus Myrothecium verrucaria (IMI 361690) containing 0.20% Silwet L-77 surfactant was found to be highly efficacious in controlling the exotic invasive weed kudzu. The mycelium can be rapidly (48–72 h) produced in several media, including an inexpensive soy flour–corn meal medium. Mycelial yields were 2, 10, and 25 g dry weight l−1 in Czapek-Dox, Richard’s V-8, and soy flour–corn meal media, respectively. Scale-up production in soy flour–corn meal medium using laboratory fermenters (10–25 l), resulted in a mycelial formulation that caused 90% mortality of naturally-occurring mature (0.9–1.0 m in height) kudzu within 48 h after application in field experiments. HPLC analyses revealed that the mycelium produced in this liquid culture contained no detectable amounts of the trichothecene mycotoxins roridin A and verrucarin A (limit of detection 2 μg ml−1). This has resulted in a safer, yet effective bioherbicidal product. We anticipate that these findings should improve the probability of EPA registration and subsequent commercial development of this bioherbicide.

Two new trichothecenes, Roridin P and Isororidin P, and two known trichothecenes, Verrucarin A and Verrucarin J, were isolated from liquid cultures of Myrothecium roridum Tale ex Fr. The stereo-structures of Roridin P and Isororidin P were established on the basis of one- and two-dimensional nuclear magnetic resonance (NMR) spectral analyses. Roridin P and Isororidin P induced morphological changes in Pyricularia oryzae, with a minimum concentration (MMCC) value of 6.2 ± 1.1 μmol/L and 7.9 ± 1.5 μmol/L.

The cytotoxicity of the extract obtained from Myrothecium roridum M10 and a characteristic (1)H signal at δH ∼8 led to the assumption that verrucarin/roridin-type compounds were present. Upscaling on rice medium led to the isolation of four new metabolites: verrucarins Y (1) and Z (6) (macrocyclic trichothecenes), bilain D (12) (a diketopiperazine derivative), and hamavellone C (14) (an unusual cyclopropyl diketone). In addition, nine known trichothecenes [verrucarin A (3), 16-hydroxyverrucarin A (5), verrucarin B (7), 16-hydroxyverrucarin B (8), verrucarin J (2), verrucarin X (4), roridin A (9), roridin L-2 (10), and trichoverritone (11)] and a bicyclic lactone [myrotheciumone A (15)] were identified. Their structures and configurations were determined by spectroscopic methods, published data, Mosher's method, and considering biosyntheses. Some trichothecenes showed motility inhibition followed by lysis of the zoospores against devastating Phytophthora nicotianae within 5 min. Compounds 2, 3, 7, and 9 also exhibited potent activities against Candida albicans and Mucor miehei.

Verrucarin A is an inhibitor of protein synthesis. In this study, we examined the inhibitory action of verrucarin A on signal molecules. Verrucarin A partially inhibited the IL-8 production of a PMA-stimulated promyelocytic leukemia cell line (HL-60 cells), and the effect was related to the inhibition of NF-κB activation at noncytotoxic concentrations. Moreover, the inhibition of mitogen activated protein (MAP) kinase by verrucarin A was especially strong with p38- and JNK-phosphorylation. The findings show a new action of verrucarin A, and it is expected that this action relaxes the signal activation in response to stress.

The efficacy of chlorine dioxide (ClO2) in detoxifying two potential bioterrorism agents, the trichothecene mycotoxins verrucarin A and roridin A, was evaluated. In the first experiment, verrucarin A (1, 5, or 10 μg) and roridin A (5 or 10 μg) were each inoculated onto square-inch sections of glass, paper, and cloth and exposed to 1000 ppm of ClO2 for either 24 or 72 h at room temperature. In the second experiment, verrucarin A and roridin A (1 or 2 ppm in water) were treated with 200, 500, or 1000 ppm ClO2 for up to 116 h at room temperature in light and dark conditions ( N = 9 per treatment for test and control). A yeast assay using Kluyveromyces marxianus was used to quantify the toxicity of verrucarin A and roridin A. Additionally, high-performance liquid chromatography was performed on selected samples. Results for the first experiment showed that ClO2 treatment had no detectable effect on either toxin. For the second experiment, both toxins were completely inactivated at all tested concentrations in as little as 2 h after treatment with 1000 ppm ClO2. For verrucarin A, an effect was seen at the 500 ppm level, but this effect was not as strong as that observed at the 1000 ppm level. Roridin A toxicity was decreased after treatment with 200 and 500 ppm ClO2, but this was not significant until the 24-h exposure time was reached. These data show that ClO2 (in solution) can be effective for detoxification of roridin A or verrucarin A at selected concentrations and exposure times.

Four macrocyclic trichothecenes, roridin A, roridin E, verrucarin A and verrucarin J, produced by the hypocrealean epibiont of Baccharis coridifolia, were evaluated for their inhibitory activity against the arenavirus Junin (JUNV), the etiological agent of Argentine hemorrhagic fever. The trichothecenes achieved a dose-dependent inhibition of JUNV multiplication at concentrations not affecting cell viability. The 50% inhibitory concentration (IC50) values determined by a virus yield inhibition assay were in the range 1.2-4.9 ng/ml. The most active compound was verrucarin J which reduced JUNV yield more than 2 log units and had a similar effect against the arenavirus Tacaribe. The trichothecenes lacked virucidal effects on JUNV virions. From time of addition and removal experiments, it can be concluded that verrucarin J inhibited a late stage in the replicative cycle of JUNV, after 5 h of adsorption. Fil: Garcia, Cybele. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Rosso, M. L.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Bertoni, María D.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Maier, Marta Silvia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina Fil: Damonte, Elsa Beatriz. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Macrocyclic trichothecenes have been frequently detected in fungi in water-damaged buildings and exhibited higher toxicity than the well-studied trichothecenes; however, the mechanism underlying their toxicity has been poorly understood. In this study, transcriptional regulation of the cytokine interleukin (IL)-8 by a macrocyclic trichothecene, verrucarin A (VA), in human monocytic THP-1 cells is reported. Consistent with previous findings, VA was 100-fold more cytotoxic than deoxynivalenol (DON), while ochratoxin A (OA) was not cytotoxic. In cells transduced with the wild-type IL-8 promoter luciferase construct, VA induced a biphasic dose response composed of an upregulation of luciferase expression at low concentrations of 0.01-1 ng/ml and a downregulation at high levels of 10 ng/ml and higher. In contrast, DON induced a sigmoid-shaped dose response with the EC50 of 11.6 ng/ml, while OA did not markedly affect the IL-8 expression. When cells were transduced with IL-8 promoter with a mutation of transcription factor nuclear factor-κB (NF-κB)-binding site, VA (1 ng/ml), DON (1000 ng/ml), and tumor necrosis factor (TNF) α (20 ng/ml)-induced luciferase expression were impaired. In addition, the NF-κB inhibitor caffeic acid phenethyl ester inhibited VA-, DON-, and TNFα-induced luciferase expression. Mutation of the CCAAT/enhancer-binding protein (CEBP) β binding site of the IL-8 promoter affected only DON-, but not VA- and TNFα-induced luciferase expression. Taken together, these results suggested that VA activated IL-8 promoter via an NF-κB-dependent, but not CEBPβ-dependent, pathway in human monocytes.

The present study was carried out to elucidate the mechanisms underlying Verrucarin A (VA)-induced cytotoxicity in human breast cancer cell line MDA-MB-231. VA inhibited the growth of MDA-MB-231 cells by induction of reactive oxygen species (ROS)-dependent mitochondrial apoptosis. Elevation of ROS production, associated with changes in Bax/Bcl-2 ratio, led to loss of mitochondrial membrane potential (Δψm) and cytochrome c release in VA-treated cells. Release of cytochrome c from mitochondria to cytosol triggered activation of caspase-3, PARP cleavage, DNA fragmentation, and finally apoptotic cell death. Furthermore, VA-induced apoptosis was accompanied by the activation of p38MAPK and inhibition of phosphorylation of EGFR as well as of Akt and ERK1/2. However, pre-treatment with n-acetyl cysteine, an ROS scavenger, and SB202190, a p38MAPK inhibitor, significantly inhibited VA-induced ROS generation, EGFR inhibition, p38MAPK activation and apoptosis. Moreover, pharmacological inhibition of EGFR and ERK1/2 significantly accelerated the VA-induced apoptosis in MDA-MB-231 cells. Collectively, these results indicate that VA-induces ROS elevation in cancer cells, which results in the activation of p38MAPK and inhibition of EGFR/Akt/ERK signaling cascade and, ultimately, cancer cell death.

Polyclonal rabbit antibodies against a conjugate synthesized through condensing BSA and disubstituted roridin A hemisuccinate allowed roridin A to be determined in solutions at a sensitivity of 0.2 ng/ml. The cross-reactivity of structural analogues—roridin A, verrucarin A, and verrucarol—amounted to 100, 2.5, and 0.03%, respectively. The data showed that these antibodies determine roridin A in an indirect heterogeneous enzyme immunoassay in cereal straw samples at a sensitivity of 20 μg/kg.

The macrocyclic trichothecenes are a group of potent protein synthesis inhibitors that have been encountered in indoor air and food as a result of infestation by the fungus Stachybotrys. To evaluate the capacity of these mycotoxins to alter immune functions, the effects of satratoxin G, H, F, roridin A, and verrucarin A on interleukin 2 (IL-2) production and viability were evaluated in a murine T-cell model. EL-4 thymoma cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin and concurrently exposed to various concentrations of the trichothecenes. Enzyme-linked immunosorbent assay (ELISA) of supernatants revealed that IL-2 concentrations at 24 and 72 h were significantly increased in cultures that were incubated in the presence of 0.5 to 1 ng/ml of satratoxin H, 1 to 5 ng/ml of isosatratoxin F, 0.1 to 0.5 ng/ml of roridin A, and 0.25 to 0.5 ng/ml of verrucarin A. However, IL-2 levels at these time points were significantly depressed when incubated in the presence of higher concentrations of satratoxin G (or =2.5 ng/ml), satratoxin H and isosatratoxin F (or =5 ng/ml), and roridin A and verrucarin A (or =1 ng/ml). Cell viability, as measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, was depressed by each of the trichothecenes in a concentration-dependent manner. MTT responses were significantly decreased by as little as 0.5 ng/ml satratoxin G, roridin A, and verrucarin A and by 2.5 ng/ml of isosatratoxin F and satratoxin H. When these data were compared to those found in EL-4 cells for the 8-ketotrichothecene vomitoxin (deoxynivalenol), a common food contaminant, the macrocyclic trichothecenes were at least 100 times more potent. The results indicate that, at low concentrations, macrocyclic trichothecenes as a group could superinduce IL-2 production even while partially decreasing cell viability, whereas higher concentrations suppressed cytokine production and were markedly cytotoxic. The capacity of these compounds to dysregulate cytokine production in a biphasic fashion may play an etiologic role in outbreaks of human illnesses associated with indoor Stachybotrys contamination.

We tested a novel colorimetric toxicity test, based on inhibition of β-galactosidase activity in the yeast Kluyveromyces marxianus , for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B 1 , ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 μg/ml, nor could aflatoxins B 1 and M 1 be detected at concentrations up to 25 μg/ml. This test should be useful for trichothecene detection and for studies of relevant interactions—both between trichothecenes themselves and between trichothecenes and other food constituents.

A novel colorimetric microbial bioassay for toxicity has been developed; it shows particular sensitivity to trichothecene mycotoxins. The assay uses inhibition of expression of β-galactosidase activity within the yeast Kluyveromyces marxianus as a sensitive toxicity indicator, cultures remaining yellow, rather than turning deep green-blue, in the presence of X-gal, a chromogenic substrate. The assay is conducted in standard microtitre plates, permitting small volumes (160 μl) and many replicates, and can be scored either automatically by a plate-reader, or by eye. Factors likely to affect the efficacy of the bioassay, including carbon source, solvents, inoculum cell density, and the use of membrane-modulating agents (MMAs), were assessed. Polymyxin B nonapeptide was the most effective toxicity-enhancing MMA tested, enabling the trichothecene mycotoxin, verrucarin A, to be detected at a concentration of about 1 ng/ml. The assay's reproducibility was examined using polymyxin B sulfate, a cheaper MMA, and another trichothecene mycotoxin, T2 toxin: reproducibility and sensitivity were better for the β-galactosidase X-gal endpoint than for an alternative chromogenic toxicity indicator, the respiratory substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

Stochastic and nonstochastic post-transcriptional gene silencing (PTGS) in Nicotiana sylvestris plants carrying tobacco class I chitinase (CHN) and beta-1,3-glucanase transgenes differs in incidence, stability, and pattern of expression. Measurements with inhibitors of RNA synthesis (cordycepin, actinomycin D, and alpha-amanitin) showed that both forms of PTGS are associated with increased sequence-specific degradation of transcripts, suggesting that increased RNA turnover may be a general feature of PTGS. The protein synthesis inhibitors cycloheximide and verrucarin A did not inhibit degradation of CHN RNA targeted for PTGS, confirming that PTGS-related RNA degradation does not depend on ongoing protein synthesis. Because verrucarin A, unlike cycloheximide, dissociates mRNA from ribosomes, our results also suggest that ribosome-associated RNA degradation pathways may not be involved in CHN PTGS.

(1)H-NMR guided fractionation of the EtOAc extract from the culture of Myrothecium gramminum (strain no. 3.1968) led to the isolation of eight cytotoxic trichothecene macrolides ( 1 - 8), all being substantially inhibitory against HepG2 (human hepatocellular carcinoma) and KB (human nasopharynyeal epidermoid tumor) cell lines with IC (50) values ranging from 2.2 to 12.2 mug/mL. Their inactivity to or weaker action on Vero cells (IC (50) values > 20 microg/mL, originated from the African green monkey kidney) highlighted preliminarily the nature of the selective cytotoxicity of the fungal metabolites against the test cancer cell lines. In addition, verrucarin A ( 1) and roridin A ( 5) were found to be potent inhibitors of the radial growth of Echinochloa crusgalli with corresponding IC (50) values of (7.96 +/- 0.31) x 10 ( - 6) and (9.18 +/- 0.44) x 10 ( - 6) M, respectively, which were more potent than that of the positive control (2,4-dichlorophenoxyacetic acid) [(9.40 +/- 0.04) x 10 ( - 5) M].

Members of the steroid receptor coactivator (SRC) family are overexpressed in numerous types of cancers. In particular, steroid receptor coactivator 3 (SRC-3) has been recognized as a critical coactivator associated with tumor initiation, progression, recurrence, metastasis, and chemoresistance where it interacts with multiple nuclear receptors and other transcription factors to enhance their transcriptional activities and facilitate cross-talk between pathways that stimulate cancer progression. Because of its central role as an integrator of growth signaling pathways, development of small molecule inhibitors (SMIs) against SRCs have the potential to simultaneously disrupt multiple signal transduction networks and transcription factors involved in tumor progression. Here, high-throughput screening was performed to identify compounds able to inhibit the intrinsic transcriptional activities of the three members of the SRC family. Verrucarin A was identified as a SMI that can selectively promote the degradation of the SRC-3 protein, while affecting SRC-1 and SRC-2 to a lesser extent and having no impact on CARM-1 and p300 protein levels. Verrucarin A was cytotoxic toward multiple types of cancer cells at low nanomolar concentrations, but not toward normal liver cells. Moreover, verrucarin A was able to inhibit expression of the SRC-3 target genes MMP2 and MMP13 and attenuated cancer cell migration. We found that verrucarin A effectively sensitized cancer cells to treatment with other anti-cancer drugs. Binding studies revealed that verrucarin A does not bind directly to SRC-3, suggesting that it inhibits SRC-3 through its interaction with an upstream effector. In conclusion, unlike other SRC SMIs characterized by our laboratory that directly bind to SRCs, verrucarin A is a potent and selective SMI that blocks SRC-3 function through an indirect mechanism.

Fumonisin B1 (FB1) is the predominant member of a family of toxic metabolites produced by several species of Fusarium and is commonly found on corn. FB1 is a potent competitive inhibitor of ceramide synthase, which catalyzes the conversion of sphinganine and sphingosine to ceramide. The resultant accumulation of free sphingoid bases and the disruption of sphingolipid metabolism is believed to be the mechanism of toxicity of the fumonisins. The objectives of this study were to determine the relative potency of analogs of FB1 to inhibit ceramide synthase and to determine whether the inhibition is specific to mycotoxins with fumonisin-like structures. Fumonisins B1, B2, B3, B4, C4, and TA toxin (a structurally similar mycotoxin produced by the tomato pathogen, Alternaria alternata f. sp. lycopersici) were approximately equipotent inhibitors. Hydrolyzed fumonisins B1, B2, and B3, which lack the tricarballylic side chains, were only 30-40% as potent as the parent toxins. N-acetylated FB1 (FA1) did not block ceramide synthase, suggesting that FA1 is nontoxic. Inhibition of ceramide synthase by fumonisin analogs did not appear to be related to the lipophilicity of the compounds, as determined by computer estimation of log P values. The ability of relatively high (10 and 100 microm) doses of other mycotoxins that bear no structural similarity to fumonisins, including aflatoxin B1, cyclopiazonic acid, beauvericin, T-2 toxin, sterigmatocystin, luteoskyrin, verrucarin A, scirpentriol, and zearalenone, to block ceramide synthase was also determined. All of the toxins tested were negative in the bioassay with the exception of fumonisins, indicating that disruption of sphingolipid metabolism is a specific cytotoxic response.

Verrucarin A (VA), a protein synthesis inhibitor, derived from the pathogen fungus Myrothecium verrucaria, inhibits growth of leukemia cell lines and activates caspases and apoptosis and inflammatory signaling in macrophages. We have investigated VA-induced growth inhibition in breast cancer cells MDA-MB-231 and T47D and, particularly, the mechanism of VA-induced apoptosis. VA treatment brought about apoptotic cell death in a dose- and time-dependent manner which was associated with chromatin condensation, cell shrinkage, nuclear fragmentation and intracellular ROS production. Mitochondrial membrane depolarization, activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax and p53 expression were observed. VA thus affects the viability of both the breast cancer cells by triggering ROS-mediated intrinsic mechanism of apoptosis.

The effects of trichothecene structure on cytokine secretion and gene expression were assessed in primary CD4 + T-cells from murine spleen. CD4 + T-cells were stimulated with concanavalin A (Con A) for 2 or 7 days in the presence of various concentrations of the trichothecenes, vomitoxin (VT or deoxynivalenol), nivalenol (NIV), 15-acetyl deoxynivalenol (15-ADON), 3-acetyl deoxynivalenol (3-ADON), T-2 toxin (T-2) and verrucarin A (Ver A). Culture supernatants were subsequently analyzed for interleukin (IL)-2, IL-4 and IL-5 by ELISA. At day 2, all trichothecenes were found to have inhibited production of IL-2, IL-4, and IL-S. However, at day 7, supernatant IL-2 was significantly increased (2–5.5-fold) in cultures containing VT, NIV, 3-ADON, and 15-ADON at 250, 250, 2500, and 1000 ng/ml doses, respectively, when compared to control Con A-stimulated cultures; significant increases in IL-2 were not observed with T-2 and Ver-A. Similarly, at day 7, IL-4 and IL-5 were significantly increased in the presence of VT (100 ng/ml), NIV (100 ng/ml), 3-ADON (1000 ng/ml), 15-ADON (500 ng/ml), T-2 (1 ng/ml), and Ver A (50 pg/ml, only IL-5) when compared to control cultures. IL production was inhibited at trichothecene concentrations exceeding the aforementioned optima. When total RNA of 2-day cultures was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) in conjunction with Southern analysis, IL-2 mRNA was also found to be superinduced by VT (50 and 100 ng/ml), NIV (50, 100 and 250 ng/ml), 3-ADON (1500 ng/ml), 15-ADON (100 ng/ml), T-2 (0.5 ng/ml) and Ver A (25, 50 and 100 pg/ml); IL-4 mRNA by VT (50 ng/ml), NIV (50 ng/ml), and Ver A (25, 50 and 100 pg/ml); IL-5 mRNA by VT (50 ng/ml); and IL-6 mRNA by 15-ADON (100 ng/ml) and Ver A (50 pg/ml). As the trichothecene concentration increased from these levels, inhibition of mRNA transcript levels was also observed for many of the interleukins. Taken together, the results suggest that trichothecenes as a group can either inhibit or superinduce both IL secretion and mRNA levels in CD4 + T-cells. Superinduction exhibited a rank order of macrocyclic > type A > type B trichothecenes and was dependent on acylation of the trichothecene nucleus.