Peroxiredoxin-2 represses NRAS-mutated melanoma cells invasion by modulating EMT markers.

The second most common mutation in melanoma occurs in NRAS oncogene, being a more aggressive disease that has no effective approved treatment. Besides, cellular plasticity limits better outcomes of the advanced and therapy-resistant patients. Peroxiredoxins (PRDXs) control cellular processes through direct hydrogen peroxide oxidation or by redox-relaying processes. Here, we demonstrated that PRDX2 could act as a modulator of multiple EMT markers in NRAS-mutated melanomas. PRDX2 knockdown lead to phenotypic changes towards invasion in human reconstructed skin and the treatment with a PRDX mimetic (gliotoxin), decreased migration in PRDX2-deficient cells. We also confirmed the favorable clinical outcome of patients expressing PRDX2 in a large primary melanoma cohort. This study contributes to our knowledge about genes involved in phenotype switching and opens a new perspective for PRDX2 as a biomarker and target in NRAS-mutated melanomas.

Characterization of Aspergillus fumigatus secretome during sublethal infection of Galleria mellonella larvae.

Introduction. The fungal pathogen Aspergillus fumigatus can induce prolonged colonization of the lungs of susceptible patients, resulting in conditions such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis.Hypothesis. Analysis of the A. fumigatus secretome released during sub-lethal infection of G. mellonella larvae may give an insight into products released during prolonged human colonisation.Methodology. Galleria mellonella larvae were infected with A. fumigatus, and the metabolism of host carbohydrate and proteins and production of fungal virulence factors were analysed. Label-free qualitative proteomic analysis was performed to identify fungal proteins in larvae at 96 hours post-infection and also to identify changes in the Galleria proteome as a result of infection.Results. Infected larvae demonstrated increasing concentrations of gliotoxin and siderophore and displayed reduced amounts of haemolymph carbohydrate and protein. Fungal proteins (399) were detected by qualitative proteomic analysis in cell-free haemolymph at 96 hours and could be categorized into seven groups, including virulence (n = 25), stress response (n = 34), DNA repair and replication (n = 39), translation (n = 22), metabolism (n = 42), released intracellular (n = 28) and cellular development and cell cycle (n = 53). Analysis of the Gallerial proteome at 96 hours post-infection revealed changes in the abundance of proteins associated with immune function, metabolism, cellular structure, insect development, transcription/translation and detoxification.Conclusion. Characterizing the impact of the fungal secretome on the host may provide an insight into how A. fumigatus damages tissue and suppresses the immune response during long-term pulmonary colonization.

Daphnia magna model for the study of mycotoxins present in food: Gliotoxin, ochratoxin A and its combination.

Mycotoxins are low molecular weight compounds present in food and feed. Although their effects on human health have been widely described, their mechanisms of action are still undefined. Gliotoxin (GTX) and ochratoxin A (OTA) are among the most dangerous mycotoxins produced by Aspergillus spp. Therefore, their toxicity was studied in the Daphnia magna model, which has high capacity to predict cytotoxicity and assess ecotoxicity, comparable to mammalian models. The study consisted of a series of tests to evaluate the effects of mycotoxins GTX, OTA and their combinations at different dilutions on Daphnia magna that were conducted according to standardized OECD 202 and 211 guidelines. The following assays were carried out: acute toxicity test, heartbeat, delayed toxicity test, reproduction, growth rate test. Reproducibility was determined by observing the offspring after 21 days of GTX exposure. In acute and delayed toxicity transcript levels of genes involved in xenobiotic metabolism (mox, gst, abcb1, and abcc5), and oxidative stress (vtg-SOD) were analyzed by qPCR. GTX showed acute toxicity and decreased heart rate in D. magna compared to OTA. On the other hand, OTA showed a delayed effect as evidenced by the immobility test. Both mycotoxins showed to increase genes involved in xenobiotic metabolism, while only the mycotoxin mixture increased oxidative stress. These results suggest that the mycotoxins tested could have negative impact on the environment and human health.

One-pot assay using a target-driven split aptamer recognition and assembly strategy for convenient and rapid detection of gliotoxin.

An aptamer targeting gliotoxin (GTX) was optimized to increase the binding affinity by approximately 20 times and achieve higher structural stability and targeting specificity. Molecular dynamics simulations were used to explore the molecular mechanism and key action sites underlying the recognition of GTX by the optimized aptamer. Subsequently, the optimized aptamer was split into two fragments and a convenient and rapid one-pot assay for GTX detection was successfully established using a target-driven split aptamer recognition and assembly strategy. The method exhibited a good linear range of 0.128 nM to 2 µM, a low detection limit of 0.07 nM, and excellent selectivity for GTX. Furthermore, the method had good accuracy and stability in real sample analysis. Therefore, the developed one-pot method provides a reliable, convenient, and cost-effective approach for the widespread application of GTX detection.

Gliotoxin elicits immunotoxicity in the early innate immune system of ducks.

Gliotoxin (GT) belongs to the epipolythiodioxopiperazine (ETP) family, which is considered a crucial virulence determinant among the secondary metabolites produced by Aspergillus fumigatus. The metabolites are commonly found in food and feed, contributing to the invasion and immune escape of Aspergillus fumigatus, thereby posing a significant threat to the health of livestock, poultry, and humans. Heterophil extracellular traps (HETs), a novel form of innate immune defense, have been documented in the chicken's innate immune systems for capturing and eliminating invading microbes. However, the effects and mechanisms of GT on the production of duck HETs in vitro remain unknown. In this study, we first confirmed the presence of HETs in duck innate immune systems and further investigated the molecular mechanism underlying GT-induced HETs release. Our results demonstrate that GT can trigger typical release of HETs in duck. The structures of GT-induced HETs structures were characterized by DNA decoration, citrullinated histones 3, and elastase. Furthermore, NADPH oxidase, glycolysis, ERK1/2 and p38 signaling pathway were found to regulate GT-induced HETs. In summary, our findings reveal that gliotoxin activates HETs release in the early innate immune system of duck while providing new insights into the immunotoxicity of GT towards ducks.

Strain heterogeneity in a non-pathogenic fungus highlights factors contributing to virulence.

Fungal pathogens exhibit extensive strain heterogeneity, including variation in virulence. Whether closely related non-pathogenic species also exhibit strain heterogeneity remains unknown. Here, we comprehensively characterized the pathogenic potentials (i.e., the ability to cause morbidity and mortality) of 16 diverse strains of Aspergillus fischeri, a non-pathogenic close relative of the major pathogen Aspergillus fumigatus. In vitro immune response assays and in vivo virulence assays using a mouse model of pulmonary aspergillosis showed that A. fischeri strains varied widely in their pathogenic potential. Furthermore, pangenome analyses suggest that A. fischeri genomic and phenotypic diversity is even greater. Genomic, transcriptomic, and metabolomic profiling identified several pathways and secondary metabolites associated with variation in virulence. Notably, strain virulence was associated with the simultaneous presence of the secondary metabolites hexadehydroastechrome and gliotoxin. We submit that examining the pathogenic potentials of non-pathogenic close relatives is key for understanding the origins of fungal pathogenicity.

Involvement of pro-inflammatory mediators and cell cycle disruption in neuronal cells induced by gliotoxin and ochratoxin A after individual and combined exposure.

Mycotoxins such as gliotoxin (GTX) and ochratoxin A (OTA) are secondary metabolites of Aspergillus and Penicillum found in food and feed. Both mycotoxins have shown to exert a detrimental effect on neuronal activity. The following study was carried out to elucidate the mechanisms by which GTX and OTA exert their toxicity. Non-differentiated SH-SY5Y neuronal-like cells were treated with GTX, OTA and their combinations to assess their cytotoxic effect using the MTT assay during 24, 48 and 72 h of exposure. Based on the results of the cytotoxic assays, cell cycle proliferation and immunological mediators were measured by determining the production of IL-6 and TNF-α using flow cytometry and ELISA, respectively. The IC50 values obtained were 1.24 and 1.35 µM when SH-SY5Y cells were treated with GTX at 48 h and 72 h, respectively. IC50 values of 8.25, 5.49 and 4.5 µM were obtained for OTA treatment at 24 h, 48 h and 72 h, respectively. The SubG0 phase increased in both treatments at 24 and 48 h. On the other hand, IL-6 and TNF-α production was increased in all mycotoxin treatments studied and was more pronounced for [GTX + OTA] after 48 h exposure. The additive and synergistic effect observed by the isobologram analysis between GTX and OTA resulted to a higher cytotoxicity which can be explained by the increased production of IL-6 and TNF-α inflammatory mediators that play an important role in the toxicity mechanism of these mycotoxins.

Marine-Fungi-Derived Gliotoxin Promotes Autophagy to Suppress Mycobacteria tuberculosis Infection in Macrophage.

The Mycobacterium tuberculosis (MTB) infection causes tuberculosis (TB) and has been a long-standing public-health threat. It is urgent that we discover novel antitubercular agents to manage the increased incidence of multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains of MTB and tackle the adverse effects of the first- and second-line antitubercular drugs. We previously found that gliotoxin (1), 12, 13-dihydroxy-fumitremorgin C (2), and helvolic acid (3) from the cultures of a deep-sea-derived fungus, Aspergillus sp. SCSIO Ind09F01, showed direct anti-TB effects. As macrophages represent the first line of the host defense system against a mycobacteria infection, here we showed that the gliotoxin exerted potent anti-tuberculosis effects in human THP-1-derived macrophages and mouse-macrophage-leukemia cell line RAW 264.7, using CFU assay and laser confocal scanning microscope analysis. Mechanistically, gliotoxin apparently increased the ratio of LC3-II/LC3-I and Atg5 expression, but did not influence macrophage polarization, IL-1ß, TNF-a, IL-10 production upon MTB infection, or ROS generation. Further study revealed that 3-MA could suppress gliotoxin-promoted autophagy and restore gliotoxin-inhibited MTB infection, indicating that gliotoxin-inhibited MTB infection can be treated through autophagy in macrophages. Therefore, we propose that marine fungi-derived gliotoxin holds the promise for the development of novel drugs for TB therapy.

Honey analysis in terms of nicotine, patulin and other mycotoxins contamination by UHPLC-ESI-MS/MS - method development and validation.

Fifty-seven samples of honey of different types and origins were screened for nicotine and nine mycotoxins (aflatoxin B1, aflatoxin B2, fusarenon X, ochratoxin A, penicillic acid, zearalenone, sterigmatocystin, gliotoxin, and patulin). The sample set consisted of monofloral, multifloral, nectar, honeydrew, cream, and artificial honey originating mainly from Poland. The physicochemical characterization of honey was performed by determining colour (by Pfund method), water content (by refractometry), total phenolics and flavonoids content (by spectrophotometry). For nicotine and mycotoxins determination a QuEChERS-based UHPLC-ESI-MS/MS method was developed and validated. Analyses were carried out in alkaline conditions to ensure patulin-methanol adduct formation and facilitate this mycotoxin detection. About 33% of tested honey samples were contaminated by nicotine or/and mycotoxins. However, the presence of mycotoxins was not related to herein evaluated physicochemical parameters of honey samples.

Protective role of CFTR during fungal infection of cystic fibrosis bronchial epithelial cells with Aspergillus fumigatus.

Lung infection with the fungus Aspergillus fumigatus (Af) is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function. We established a fungal epithelial co-culture model to examine the impact of Af infection on CF bronchial epithelial barrier function using Af strains 10AF and AF293-GFP, and the CFBE41o- cell line homozygous for the F508del mutation with (CF+CFTR) and without (CF) normal CFTR expression. Following exposure of the epithelial surface to Af conidia, formation of germlings (early stages of fungal growth) was detected after 9-12 hours and hyphae (mature fungal growth) after 12-24 hours. During fungal morphogenesis, bronchial epithelial cells showed signs of damage including rounding, and partial detachment after 24 hours. Fluorescently labeled conidia were internalized after 6 hours and more internalized conidia were observed in CF compared to CF+CFTR cells. Infection of the apical surface with 10AF conidia, germlings, or hyphae was performed to determine growth stage-specific effects on tight junction protein zona occludens protein 1 (ZO-1) expression and transepithelial electrical resistance (TER). In response to infection with conidia or germlings, epithelial barrier function degraded time-dependently (based on ZO-1 immunofluorescence and TER) with a delayed onset in CF+CFTR cell monolayers and required viable fungi and apical application. Infection with hyphae caused an earlier onset and faster rate of decline in TER compared to conidia and germlings. Gliotoxin, a major Af virulence factor, caused a rapid decline in TER and induced a transient chloride secretory response in CF+CFTR but not CF cells. Our findings suggest growth and internalization of Af result in deleterious effects on bronchial epithelial barrier function that occurred more rapidly in the absence of CFTR. Bronchial epithelial barrier breakdown was time-dependent and morphotype-specific and mimicked by acute administration of gliotoxin. Our study also suggests a protective role for CFTR by turning on CFTR-dependent chloride transport in response to gliotoxin, a mechanism that will support mucociliary clearance, and could delay the loss of epithelial integrity during fungal development in vivo.

Molecular characterization of gliotoxin-producing Aspergillus fumigatus in dairy cattle feed.

Background and Aim: Several strains of Aspergillus fumigatus produce mycotoxins that affect the health and productivity of dairy cattle, and their presence in dairy cattle feed is a serious concern. This study aimed to determine the densities of A. fumigatus and gliotoxin in commercial dairy feed. Materials and Methods: More than 60 dairy feed samples were examined for fungal contamination, specifically for A. fumigatus, using phenotypic approaches and DNA sequencing of the internal transcribed spacer (ITS) and ß-tubulin regions. Thin-layer chromatography and high-performance liquid chromatography (HPLC) were used to assess gliotoxin production in A. fumigatus. Real-time polymerase chain reaction (RT-PCR) was used to investigate the expression of gliZ, which was responsible for gliotoxin production. High-performance liquid chromatography was used to detect gliotoxin in feed samples. Results: Aspergillus was the most commonly identified genus (68.3%). Aspergillus fumigatus was isolated from 18.3% of dairy feed samples. Only four of the 11 A. fumigatus isolates yielded detectable gliotoxins by HPLC. In total, 7/11 (43.7%) feed samples tested had gliotoxin contamination above the threshold known to induce immunosuppressive and apoptotic effects in vitro. The HPLC-based classification of isolates as high, moderate, or non-producers of gliotoxin was confirmed by RT-PCR, and the evaluation of gliZ expression levels corroborated this classification. Conclusion: The identification of A. fumigatus from animal feed greatly depended on ITS and ß-tubulin sequencing. Significant concentrations of gliotoxin were found in dairy cattle feed, and its presence may affect dairy cow productivity and health. Furthermore, workers face contamination risks when handling and storing animal feed.

Vacuoles and peroxisomes are involved in Aspergillus fumigatus gliotoxin production and self-protection.

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. Peroxisomes are also required for proper GT production and self-defense. The Mitogen-Activated Protein (MAP) kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. Our work emphasizes the importance of dynamic compartmentalization of cellular events for GT production and self-defense.

Aspergillus fumigatus Supernatants Disrupt Bronchial Epithelial Monolayers: Potential Role for Enhanced Invasion in Cystic Fibrosis.

Aspergillus fumigatus is the most commonly isolated fungus in chronic lung diseases, with a prevalence of up to 60% in cystic fibrosis patients. Despite this, the impact of A. fumigatus colonisation on lung epithelia has not been thoroughly explored. We investigated the influence of A. fumigatus supernatants and the secondary metabolite, gliotoxin, on human bronchial epithelial cells (HBE) and CF bronchial epithelial (CFBE) cells. CFBE (F508del CFBE41o-) and HBE (16HBE14o-) trans-epithelial electrical resistance (TEER) was measured following exposure to A. fumigatus reference and clinical isolates, a gliotoxin-deficient mutant (ΔgliG) and pure gliotoxin. The impact on tight junction (TJ) proteins, zonula occludens-1 (ZO-1) and junctional adhesion molecule-A (JAM-A) were determined by western blot analysis and confocal microscopy. A. fumigatus conidia and supernatants caused significant disruption to CFBE and HBE TJs within 24 h. Supernatants from later cultures (72 h) caused the greatest disruption while ΔgliG mutant supernatants caused no disruption to TJ integrity. The ZO-1 and JAM-A distribution in epithelial monolayers were altered by A. fumigatus supernatants but not by ΔgliG supernatants, suggesting that gliotoxin is involved in this process. The fact that ΔgliG conidia were still capable of disrupting epithelial monolayers indicates that direct cell-cell contact also plays a role, independently of gliotoxin production. Gliotoxin is capable of disrupting TJ integrity which has the potential to contribute to airway damage, and enhance microbial invasion and sensitisation in CF.

Gliotoxin and related metabolites as zinc chelators: implications and exploitation to overcome antimicrobial resistance.

Antimicrobial resistance (AMR) is a major global problem and threat to humanity. The search for new antibiotics is directed towards targeting of novel microbial systems and enzymes, as well as augmenting the activity of pre-existing antimicrobials. Sulphur-containing metabolites (e.g., auranofin and bacterial dithiolopyrrolones [e.g., holomycin]) and Zn2+-chelating ionophores (PBT2) have emerged as important antimicrobial classes. The sulphur-containing, non-ribosomal peptide gliotoxin, biosynthesised by Aspergillus fumigatus and other fungi exhibits potent antimicrobial activity, especially in the dithiol form (dithiol gliotoxin; DTG). Specifically, it has been revealed that deletion of the enzymes gliotoxin oxidoreductase GliT, bis-thiomethyltransferase GtmA or the transporter GliA dramatically sensitise A. fumigatus to gliotoxin presence. Indeed, the double deletion strain A. fumigatus ΔgliTΔgtmA is especially sensitive to gliotoxin-mediated growth inhibition, which can be reversed by Zn2+ presence. Moreover, DTG is a Zn2+ chelator which can eject zinc from enzymes and inhibit activity. Although multiple studies have demonstrated the potent antibacterial effect of gliotoxin, no mechanistic details are available. Interestingly, reduced holomycin can inhibit metallo-ß-lactamases. Since holomycin and gliotoxin can chelate Zn2+, resulting in metalloenzyme inhibition, we propose that this metal-chelating characteristic of these metabolites requires immediate investigation to identify new antibacterial drug targets or to augment the activity of existing antimicrobials. Given that (i) gliotoxin has been shown in vitro to significantly enhance vancomycin activity against Staphylococcus aureus, and (ii) that it has been independently proposed as an ideal probe to dissect the central 'Integrator' role of Zn2+ in bacteria - we contend such studies are immediately undertaken to help address AMR.

Quercetin alleviates gliotoxin-induced duckling tissue injury by inhibiting oxidative stress, inflammation and increasing heterophil extracellular traps release.

Aspergillus fumigatus causes aspergillosis with high morbidity and mortality in the duck industry. As a vital virulence factor produced by A. fumigatus, gliotoxin (GT) is widely present in food and feed, threatening duck industry and human health. Quercetin is a polyphenol flavonoid compound from natural plants with anti-inflammatory and antioxidant functions. However, the effects of quercetin on ducklings with GT poisoning are unknown. The model of ducklings with GT poisoning was established, and the protective effects and molecular mechanisms of quercetin on ducklings with GT poisoning were investigated. Ducklings were divided into control, GT, and quercetin groups. A model of GT (2.5 mg/kg) poisoning in ducklings was successfully established. Quercetin protected GT-induced liver and kidney functions and alleviated GT-induced alveolar wall thickening in lungs, cell fragmentation, and inflammatory cell infiltration in liver and kidney. Quercetin decreased malondialdehyde (MDA) and increased superoxide dismutase (SOD) and catalase (CAT) after GT treatment. Quercetin significantly reduced GT-induced mRNA expression levels of inflammatory factors. Furthermore, quercetin increased GT-reduced heterophil extracellular traps (HETs) in serum. These results indicated that quercetin protected ducklings against GT poisoning by inhibiting oxidative stress, inflammation and increasing HETs release, which confirms the potential applicability of quercetin in treating GT-induced duckling poisoning.

Exposure of Aspergillus fumigatus to Klebsiella pneumoniae Culture Filtrate Inhibits Growth and Stimulates Gliotoxin Production.

Aspergillus fumigatus is an opportunistic fungal pathogen capable of inducing chronic and acute infection in susceptible patients. A. fumigatus interacts with numerous bacteria that compose the microbiota of the lung, including Pseudomonas aeruginosa and Klebsiella pneumoniae, both of which are common isolates from cystic fibrosis sputum. Exposure of A. fumigatus to K. pneumoniae culture filtrate reduced fungal growth and increased gliotoxin production. Qualitative proteomic analysis of the K. pneumoniae culture filtrate identified proteins associated with metal sequestering, enzymatic degradation and redox activity, which may impact fungal growth and development. Quantitative proteomic analysis of A. fumigatus following exposure to K. pneumoniae culture filtrate (25% v/v) for 24 h revealed a reduced abundance of 1,3-beta-glucanosyltransferase (-3.97 fold), methyl sterol monooxygenase erg25B (-2.9 fold) and calcium/calmodulin-dependent protein kinase (-4.2 fold) involved in fungal development, and increased abundance of glutathione S-transferase GliG (+6.17 fold), non-ribosomal peptide synthase GliP (+3.67 fold), O-methyltransferase GliM (+3.5 fold), gamma-glutamyl acyltransferase GliK (+2.89 fold) and thioredoxin reductase GliT (+2.33 fold) involved in gliotoxin production. These results reveal that exposure of A. fumigatus to K. pneumoniae in vivo could exacerbate infection and negatively impact patient prognosis.

Gliotoxin, a natural product with ferroptosis inducing properties.

As one of the mycotoxins produced by Aspergillus fumigatus, gliotoxin has a variety of pharmacological effects, such as anti-tumor, antibacterial, immunosuppressive. Antitumor drugs induce tumor cell death in several forms, including apoptosis, autophagy, necrosis and ferroptosis. Ferroptosis is a recently identified unique form of programmed cell death characterized by iron-dependent accumulation of lethal lipid peroxides, which induces cell death. A large amount of preclinical evidence suggests that ferroptosis inducers may enhance the sensitivity of chemotherapy and the induction of ferroptosis may be an effective therapeutic strategy to prevent acquired drug resistance. In our study, gliotoxin was characterized as a ferroptosis inducer and showed strong anti-tumor activity with IC50 of 0.24 µM and 0.45 µM in H1975 and MCF-7 cells at 72 h, respectively. Gliotoxin may provide a new natural template for the designing of ferroptosis inducers.

6-Heterocyclic carboxylic ester derivatives of gliotoxin lead to LSD1 inhibitors in gastric cancer cells.

Gliotoxin is a representative compound of the epipolythiodioxopiperazine (ETP) class of fungal metabolites. Histone Lysine Specific Demethylase 1 (LSD1) is highly expressed in a variety of cancers. Herein, a series of 6-heterocyclic carboxylic ester derivatives of gliotoxin was designed and synthesized as new LSD1 inhibitors and their biological evaluations in human gastric MGC-803 and HGC-27 cells were carried out. All of the derivatives effectively suppressed the enzymatic activities of LSD1. In particular, compound 4e exhibited excellent LSD1 inhibition with IC50 = 62.40 nM, as well as anti-proliferation against MGC-803 and HGC-27 cells with IC50 values of 0.31 µM and 0.29 µM, respectively. 4e also had a remarkable capacity to inhibit the colony formation, suppress migration and induce the apoptosis of these two cancer cell lines. In sum, our findings identified and characterized the 6-heterocyclic carboxylic ester derivatives of gliotoxin as potent and cellular active LSD1 inhibitors, which may provide a novel chemotype of LSD1 inhibitors for gastric cancer treatment.

Gliotoxin Induced Ferroptosis by Downregulating SUV39H1 Expression in Esophageal Cancer Cells.

BACKGROUND: Gliotoxin, a secondary metabolite isolated from marine-derived Aspergillus fumigatus, has demonstrated anti-tumor properties in several cancers. Ferroptosis, a recently discovered type of programmed cell death that depends on the accumulation of iron and lipid peroxides, participates in the occurrence and development of various diseases, including cancer. A recent patent, US20200383943, has suggested that the promotion of ferroptosis is a method of cancer treatment. Therefore, the development of drugs that induce ferroptosis in cancer cells would constitute a novel therapeutic approach. OBJECTIVE: Gliotoxin is a natural compound which has exhibited anti-tumor properties in multiple cancers, however, studies of the effect of gliotoxin on esophageal cancer are lacking. Although cancer treatment has shown great progress, including traditional surgery, chemotherapy, radiotherapy, and immunotherapy, the prognosis of esophageal cancer is still poor. Therefore, the development of new treatment approaches for esophageal cancer is necessary. METHODS: The effects of gliotoxin on esophageal cancer cells were determined by functional assays, such as CCK-8, wound healing and transwell assays. We used online tools to predict the target genes of gliotoxin, followed by further verification using Western blotting assays. To assess the role of gliotxin in inducing ferroptosis in esophageal cancer, we detected characteristics associated with ferroptosis including ROS, MDA, GSH and Fe2+. RESULTS: Using online tools SEA and SwissTargetPrediction, we predicted that SUV39H1 was the gliotoxin target gene. Furthermore, in esophageal cancer tissues, SUV39H1 was expressed at higher levels than in normal tissues, while in patients with Esophageal Squamous Cell Carcinoma (ESCC), high expression levels of SUV39H1 indicated a poor prognosis. In vitro, we observed that gliotoxin increased ESCC cell death and inhibited cell migration. We treated ESCC cells with pan-caspase inhibitor Z-VAD-FMK or ferroptosis inhibitors, including Fer-1 and DFO. Our results showed that Fer-1 and DFO reduced the toxic effects of gliotoxin, while Z-VAD-FMK did not. Furthermore, gliotoxin treatment reduced tumor weight and volume in the xenograft tumor mouse model. CONCLUSION: In summary, our findings indicate that gliotoxin downregulated SUV39H1 expression in ESCC cells and induced ferroptosis, suggesting a novel natural therapy for ESSC.

Combination Treatment Using Pyruvate Kinase M2 Inhibitors for the Sensitization of High Density Triple-negative Breast Cancer Cells.

BACKGROUND/AIM: Few studies have examined the correlation between pyruvate kinase M2 (PKM2) overexpression and triple-negative breast cancer (TNBC). TNBC is considered incurable with the currently available treatments, highlighting the need for alternative therapeutic targets. MATERIALS AND METHODS: PKM2 expression was examined immunohistochemically in human breast tumor samples. Furthermore, we studied the effect of three PKM2 inhibitors (gliotoxin, shikonin, and compound 3K) in the MDA-MB-231 TNBC cell line. RESULTS: PKM2 overexpression correlates with TNBC. Interestingly, most TNBC tissues showed increased levels of PKM2 compared to those of receptor-positive breast cancer tissues. This suggests that PKM2 overexpression is an important factor in the development of TNBC. MDA-MB-231 TNBC cells are resistant to anticancer drugs, such as vincristine (VIC) compared to other cancer cells. We found that the recently developed PKM2 inhibitor gliotoxin sensitized MDA-MB-231 cells at a relatively low dose to the same extent as the known PKM2 inhibitor shikonin, suggesting that PKM2 inhibitors could be an effective treatment for TNBC. Detailed sensitization mechanisms were also analyzed. Both gliotoxin and shikonin highly increased late apoptosis in MDA-MB-231 cells, as revealed by annexin V staining. However, MDA-MB-231 cells with high cellular density inhibited the sensitizing effect of PKM2 inhibitors; therefore, we investigated ways to overcome this inhibitory effect. We found that gliotoxin+shikonin co-treatment highly increased toxicity in MDA-MB-231 cells with high density, whereas either VIC+gliotoxin or VIC+shikonin were not effective. Thus, combination therapy with various PKM2 inhibitors may be more effective than combination therapy with anticancer drugs. Gliotoxin+shikonin co-treatment did not increase S or G2 arrest in cells, suggesting that the co-treatment showed a high increase in apoptosis without S or G2 arrest. We confirmed that another recently developed PKM2 inhibitor compound 3K had similar mechanisms of sensitizing MDA-MB-231 cells, suggesting that PKM2 inhibitors have similar sensitization mechanisms in TNBC. CONCLUSION: PKM2 is a regulator of the oncogenic function of TNBC, and combination therapy with various PKM2 inhibitors may be effective for high-density TNBC. Targeting PKM2 in TNBC lays the foundation for the development of PKM2 inhibitors as promising anti-TNBC agents.