Identification of Gliotoxin isolated from marine fungus as a new pyruvate kinase M2 inhibitor.

Pyruvate kinase M2 (PKM2) functions as an important rate-limiting enzyme of aerobic glycolysis that is involved in tumor initiation and progression. However, there are few studies on effective PKM2 inhibitors. Gliotoxin is a marine-derived fungal secondary metabolite with multiple biological activities, including immunosuppression, cytotoxicity, and et al. In this study, we found that Gliotoxin directly bound to PKM2 and inhibited its glycolytic activity in a dose-dependent manner accompanied by the decreases in glucose consumption and lactate production in the human glioma cell line U87. Moreover, Gliotoxin suppressed tyrosine kinase activity of PKM2, leading to a dramatic reduction in Stat3 phosphorylation in U87 cells. Furthermore, Gliotoxin suppressed cell viability in U87 cells, and cytotoxicity of Gliotoxin on U87 cells was obviously augmented under hypoxia condition compared to normal condition. Finally, Gliotoxin was demonstrated to induce cell apoptosis of U87 cells and synergize with temozolomide. Our findings identify Gliotoxin as a new PKM2 inhibitor with anti-tumor activity, which lays the foundation for the development of Gliotoxin as a promising anti-tumor drug in the future.

Targeting Nuclear NOTCH2 by Gliotoxin Recovers a Tumor-Suppressor NOTCH3 Activity in CLL.

NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH γ-secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the NOTCH2/FCER2 (CD23) axis together with concomitant upregulation of the NOTCH3/NR4A1 axis. In contrast, RO4929097 downregulated the NOTCH3/NR4A1 axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NFκB, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.

MRI-guided intracerebral convection-enhanced injection of gliotoxins to induce focal demyelination in swine.

Demyelinating disorders such as multiple sclerosis (MS) or transverse myelitis are devastating neurological conditions with no effective cure. Prevention of myelin loss or restoration of myelin are key for successful therapy. To investigate the disease and develop cures animal models with good clinical relevance are essential. The goal of the current study was to establish a model of focal demyelination in the brain of domestic pig using MRI-guided gliotoxin delivery. The rationale for developing a new myelin disease model in the domestic pig was based on the fact that the brain in pigs is anatomically and histologically much more similar to that of humans compared to the rodent brain. For MRI-assisted gliotoxin injection, eight 30 kg pigs were subjected to treatment with lysolecithin (20, 30 mg/ml); or with ethidium bromide (0.0125, 0.05, 0.2 mg/ml). Animals were placed in an MRI scanner for intraparenchymal targeting of gliotoxin into the corona radiata (250 µl over 1h), with real-time monitoring of toxin distribution on T1 scans and monitoring of lesion evolution over seven days using both T1 and T2 scans. After the last MRI, animals were transcardially perfused and brains were processed for histological and immunofluorescent analysis. Gadolinium-enhanced T1 MRI during injection demonstrated biodistribution of the contrast (as a surrogate marker for toxin distribution) and its diffusion through the brain parenchyma. Lesion induction was confirmed on T2-weighted MRI and histopathology, thus enabling the establishment of optimal doses of gliotoxins. To conclude, MRI-guided focal demyelination in swine is accurate and provides real-time confirmation of gliotoxin, thus facilitating placement of focal lesions with high precision. This new model of focal demyelination can be used for further investigation and development of novel therapeutic approaches.

Effects of the Combination of Gliotoxin and Adriamycin on the Adriamycin-Resistant Non-Small-Cell Lung Cancer A549 Cell Line.

Acquired drug resistance constitutes an enormous hurdle in cancer treatment, and the search for effective compounds against resistant cancer is still advancing. Marine organisms are a promising natural resource for the discovery and development of anticancer agents. In this study, we examined whether gliotoxin (GTX), a secondary metabolite isolated from marine-derived Aspergillus fumigatus, inhibits the growth of adriamycin (ADR)-resistant non-small-cell lung cancer (NSCLC) cell lines A549/ADR. We investigated the effects of GTX on A549/ADR cell viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the induction of apoptosis in A549/ADR cells treated with GTX via fluorescence-activated cell sorting analysis, Hoechst staining, annexin V/propidium iodide staining, tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, and western blotting. We found that GTX induced apoptosis in A549/ADR cells through the mitochondria-dependent pathway by disrupting mitochondrial membrane potential and activating p53, thereby increasing the expression levels of p21, p53 upregulated modulator of apoptosis (PUMA), Bax, cleaved poly (ADP-ribose) polymerase (PARP), and cleaved caspase-9. More importantly, we discovered that GTX works in conjunction with ADR to exert combinational effects on A549/ADR cells. In conclusion, our results suggest that GTX may have promising effects on ADR-resistant NSCLC cells by inducing mitochondria-dependent apoptosis and through the combined effects of sequential treatment with ADR.

Disseminated aspergillosis in an immunocompetent patient with detectable bis(methylthio)gliotoxin and negative galactomannan / Aspergilosis diseminada en un paciente inmunocompetente con bis(metiltio)gliotoxina detectable y galactomanano negativo

Background. Disseminated invasive aspergillosis is an exceptional finding in immunocompetent hosts. As in immunocompromised patients, it has high mortality rates. Early diagnostic methods are required in order to properly manage the patient. Bis(methylthio)gliotoxin (bmGT) is a novel biomarker, useful in onco-hematological patients. Case report. A 70-year-old male, with non-insulin dependent type II diabetes mellitus and a past surgery history of aortic valve replacement with coronary by-pass five years ago, was seen in the emergency department with blurred vision. Three days later, endogen endophthalmitis was diagnosed in the ophthalmology clinic. During admission for the vitrectomy, he suffered an ischemia of the right lower limb. A thoracic computed tomography revealed a mycotic aneurysm of the ascending thoracic aorta and parietal thrombus. The ascending aorta was replaced and abundant brittle material of infectious appearance, found between the aortic valve graft and the aneurysm, was removed. Aspergillus fumigatus sensu stricto grew in both vitreous and aorta cultures. BmGT was detected in two serum samples obtained prior to intravenous antifungal treatment, which was then reduced after voriconazole treatment was started. Conclusions. Disseminated invasive aspergillosis is a severe disease regardless of the immune status of the patient. This case report suggests that bmGT could be a suitable early diagnostic biomarker, not only in neutropenic patients, but also in immunocompetent hosts (AU)

Antecedentes. La aspergilosis diseminada invasiva es un hallazgo excepcional en pacientes inmunocompetentes, y al igual que en los pacientes inmunodeficientes, alcanza valores de mortalidad elevados. Para el correcto manejo del paciente son necesarios métodos diagnósticos precoces. La bis(metiltio)gliotoxina es un nuevo biomarcador de gran utilidad en pacientes oncohematológicos. Caso clínico. Varón de 70 años de edad con diabetes mellitus tipo II no dependiente de insulina y antecedente de recambio valvular aórtico con by-pass coronario cinco años antes, que acude al Servicio de Urgencias por visión borrosa. Tres días después se le diagnosticó endoftalmitis endógena en la consulta de Oftalmología. Durante su ingreso para la vitrectomía presentó una isquemia del miembro inferior derecho. La tomografía computarizada de tórax reveló un aneurisma micótico en la aorta torácica ascendente y un trombo parietal. Se reemplazó la aorta ascendente y se eliminó abundante material friable de aspecto infeccioso entre la prótesis valvular aórtica y el aneurisma. En los cultivos de humor vítreo y aorta creció Aspergillus fumigatus sensu stricto. Se detectó bis(metiltio)gliotoxina en dos muestras de suero obtenidas antes del tratamiento antifúngico intravenoso, marcador que disminuyó tras comenzar el tratamiento con voriconazol. Conclusiones. La aspergilosis diseminada invasiva es una enfermedad grave independientemente del estado inmune del paciente. Este caso clínico evidencia que la bis(metiltio)gliotoxina podría ser un marcador diagnóstico precoz no solo en pacientes neutropénicos, sino también en huéspedes inmunocompetentes (AU)

Polymer functionalized single walled carbon nanotubes mediated drug delivery of gliotoxin in cancer cells.

During recent years, significant development has been achieved in carbon nanotube conjugated with polymer system for drug delivery system (DDS). In the present study, we have prepared functionalized single walled carbon nanotube conjugated with chitooligosaccharide (f-SWNT-COS) as a Drug Delivery System. In addition, drug Gliotoxin (GTX) and targeting molecules (Lysozyme, p53 and Folic acid) have been incorporated into f-SWNT-COS. f-SWNTs-COS-GTX-p53, f-SWNTs-COS-GTX-lysozyme, f-SWNTs-COS-GTX-FA have been physiochemically characterized for DDS. FT-IR, SEM and TEM analysis confirmed the formation of chemical interaction and polymer coating. FT-IR result clearly confirmed the interaction between f-SWNT and COS. The effective drug release was monitored against cervical cancer (HeLa) cells and Breast Cancer (MCF-7) cells and it was found that all the three drug delivery systems showed significant cytotoxicity. f-SWNTs-COS-GTX-p53 delivery vehicle and its effective cytotoxicity on HeLa cells was further checked with fluorescent activated cell sorter analysis. Our results suggest that the f-SWNTs-COS-GTX-p53 is the most effective delivery vehicle with a controlled release and enhanced cytotoxicity rendered through apoptosis in human cervical cancer (HeLa) cells. These systems can further be used for the delivery of other commercially available anti cancer drugs as well.

Individual and combined effects of mycotoxins from typical indoor moulds.

The mycotoxins patulin, gliotoxin and sterigmatocystin can be produced by common indoor moulds and enter the human body via inhalation of mycotoxin containing spores and particulates. There are various studies about the individual effects of these mycotoxins, but a lack of knowledge about their effects in mixtures. The aim of this study was to evaluate combined effects on the singe celled organism Tetrahymena pyriformis. Using the MIXTOX model (EU project NOMIRACLE) synergistic or antagonistic effects with dose level deviation or dose ratio dependent deviation were analyzed. The most toxic compound gliotoxin (EC50 0.37 µM) and patulin (EC50 9.3 µM) as shown by the MIXTOX model acted synergistic, caused by similar mode of actions. Within the combination with sterigmatocystin (maximum inhibition of 45% at 12.5 µM) antagonistic effects were observed with switch to synergism if the toxicity of the mixture is mainly caused by sterigmatocystin. In the end the MIXTOX model was applicable for the prediction of combined effects of toxic compounds.

Cellular targeting of the apoptosis-inducing compound gliotoxin to fibrotic rat livers.

Liver fibrosis is associated with proliferation of hepatic stellate cells (HSCs) and their transformation into myofibroblastic cells that synthesize scar tissue. Several studies indicate that induction of apoptosis in myofibroblastic cells may prevent fibrogenesis. Gliotoxin (GTX) was found to induce apoptosis of hepatic cells and caused regression of liver fibrosis. However, the use of apoptosis-inducing drugs may be limited due to lack of cell specificity, with a risk of severe adverse effects. In previous studies, we found that mannose-6-phosphate-modified human serum albumin (M6P-HSA) selectively accumulated in liver fibrogenic cells. The aim of this study therefore was to couple GTX to M6P-HSA and test its pharmacological effects in vitro and in rats with liver fibrosis. The conjugate GTX-M6P-HSA bound specifically to HSCs and reduced their viability. Apoptosis was induced in cultures of human hepatic myofibroblasts (hMFs) and in liver slices obtained from rats with liver fibrosis. In vivo treatment with GTX or GTX-M6P-HSA in bile duct ligated rats revealed a significant decrease in alpha-smooth muscle actin mRNA levels and a reduced staining for this HSC marker in fibrotic livers. In addition, although GTX also affected hepatocytes, GTX-M6P-HSA did not significantly affect other liver cells. In conclusion, we developed an HSC-specific compound that induced apoptosis in human hMFs, rat HSCs, and in fibrotic liver slices. In vivo, both GTX and GTX-M6P-HSA attenuated the number of activated HSCs, but GTX also affected hepatocytes. This study shows that cell-selective delivery of the apoptosis-inducing agent GTX is feasible in fibrotic livers.

Mechanism of action of the antifibrogenic compound gliotoxin in rat liver cells.

Gliotoxin has been shown to promote a reversal of liver fibrosis in an animal model of the disease although its mechanism of action in the liver is poorly defined. The effects of gliotoxin on activated hepatic stellate cells (HSCs) and hepatocytes have therefore been examined. Addition of gliotoxin (1.5 microM) to culture-activated HSCs resulted in its rapid accumulation, resulting in increased levels of glutathione and apoptosis without any evidence of oxidative stress. In contrast, although hepatocytes also rapidly sequestered gliotoxin, cell death only occurred at high (50-microM) concentrations of gliotoxin and by necrosis. At high concentrations, gliotoxin was metabolized by hepatocytes to a reduced (dithiol) metabolite and glutathione was rapidly oxidized. Fluorescent dye loading experiments showed that gliotoxin caused oxidative stress in hepatocytes. Antioxidants--but not thiol redox active compounds--inhibited both oxidative stress and necrosis in hepatocytes. In contrast, HSC apoptosis was not affected by antioxidants but was potently abrogated by thiol redox active compounds. The adenine nucleotide transporter (ANT) is implicated in mitochondrial-dependent apoptosis. HSCs expressed predominantly nonliver ANT isoform 1, and gliotoxin treatment resulted in a thiol redox-dependent alteration in ANT mobility in HSC extracts, but not hepatocyte extracts. In conclusion, these data suggest that gliotoxin stimulates the apoptosis of HSCs through a specific thiol redox-dependent interaction with the ANT. Further understanding of this mechanism of cell death will aid in finding therapeutics that specifically stimulate HSC apoptosis in the liver, a promising approach to antifibrotic therapy.

Targeting hepatic stellate cells for cell-specific treatment of liver fibrosis.

Since hepatic stellate cells (HSC) play a crucial role in the development of liver fibrosis, this cell is the major target for anti-fibrotic drugs. Most of the experimental drugs that influenced the HSC activity showed however low efficacy in vivo. Either a low uptake of the compounds in the cells that cause disease might account for this lack of effect, or side-effects in other cells may limit the dosage of the drugs. These side-effects may even counteract the beneficial effects. Therefore a selective delivery of drugs to the HSC may comprise a promising new way to improve liver fibrosis. The targeting to HSC has become a feasible option, because albumin-based carriers have been developed that preferentially distribute to HSC in fibrotic rat livers. In addition to the targeting of drugs, also the selective delivery of genes to HSC in fibrotic livers is of interest for therapeutic purposes and a start is made in this respect. The present review discusses the drugs to be targeted to HSC and summarizes some of the problems encountered during this novel strategy in the treatment of liver fibrosis.

Nuclear factor-kappa B activity and intestinal inflammation in dextran sulphate sodium (DSS)-induced colitis in mice is suppressed by gliotoxin.

In acute DSS-induced colitis nuclear factor (NF)-kappaB-dependent inflammatory cytokines including IL-1 and tumour necrosis factor-alpha (TNF-alpha) are up-regulated. Here we examined the effects of gliotoxin, a fungal metabolite known to inhibit NF-kappaB activity, on cytokine production by a mouse cell system in vitro and on intestinal inflammation and NF-kappaB activation in vivo. In vitro gliotoxin decreased TNF-alpha gene expression and protein production by RAW-264.7 mouse macrophage-like cells stimulated with lipopolysaccharide. In vivo, gliotoxin treatment of mice was begun on day 3 of 5% DSS application dissolved in the drinking water and continued until day 8. Gliotoxin treatment dose-dependently down-regulated colonic inflammation as assessed histologically and in parallel there was a suppression of colonic TNF-alpha and IL-1alpha mRNA expression on day 8 as analysed by semiquantitative reverse transcriptase-polymerase chain reaction (P < 0.01). Furthermore, colonic NF-kappaB DNA-binding activity was increased in DSS-induced colitis and was suppressed by gliotoxin. These results demonstrate the essential role of NF-kappaB in DSS-induced colitis and indicate a molecular approach to the treatment of intestinal inflammatory disorders.

Inhibition of corneal inflammation by the topical use of Ras farnesyltransferase inhibitors: selective inhibition of macrophage localization.

PURPOSE: Ras farnesyltransferase inhibitors are known to block the membrane translocalization of oncogenic Ras protein. They inhibit the cytoplasmic mitogen-activated protein kinase signaling cascade related to Ras protein. Thus far, Ras farnesyltransferase inhibitors have been exclusively regarded with the anticancer drugs. The object of this study was to elucidate the role of Ras farnesyltransferase inhibitors on the corneal opacity induced by an inflammatory stimulus. METHODS: We used a cauterization-induced corneal inflammation model. The central corneas of BALB/c mice were cauterized with silver nitrate (1 mm in diameter). Ras farnesyltransferase inhibitors, either manumycin or gliotoxin eye drops (each drug dissolved in balanced salt solution [BSS] at concentrations of 1 mM), were topically delivered to the cauterized cornea every 8 hours; BSS eye drops were used as a control. Clinical signs such as corneal edema, opacity, and corneal neovascularization, which are major causes of visual disturbance, were then examined 96 hours after the cauterization. The corneal edema and opacity were clinically scored under a stereoscopic microscope. The corneal neovascularization was evaluated by the length of the blood vessels from the limbus and the sum of extension central angle of vascularized limbus. Furthermore, the corneas were examined histologically, and the phenotypes of the cornea-infiltrating cells were analyzed by flow cytometry. RESULTS: The control corneas showed prominent edema, neovascularization, and opacity. Histologic analysis revealed corneal epithelial and endothelial cell loss and a large amount of inflammatory cell infiltration into the corneal stroma. Flow cytometric analysis revealed that most of the infiltrating cells were neutrophils and macrophages. In contrast, the degree of corneal edema, neovascularization, and opacity was significantly less in the manumycin- or gliotoxin-treated corneas than in the control corneas. Histologically, the manumycin- and gliotoxin-treated corneas showed minimum edema and good epithelialization. Flow cytometric analysis showed corneal infiltration of macrophages to be selectively and clearly inhibited. Neither manumycin nor gliotoxin produced any side effects in the noncauterized normal cornea either clinically or histologically. CONCLUSIONS: Ras proteins play an important role in cauterization-induced corneal inflammation and the opacity it induces. Ras farnesyltransferase inhibitors thus have a great potential for improving the treatment of corneal opacity induced by a corneal inflammatory stimulus.