Antifungal drug resistance in Candida: a special emphasis on amphotericin B.

Invasive fungal infections in humans caused by several Candida species, increased considerably in immunocompromised or critically ill patients, resulting in substantial morbidity and mortality. Candida albicans is the most prevalent species, although the frequency of these organisms varies greatly according to geographic region. Infections with C. albicans and non-albicans Candida species have become more common, especially in the past 20 years, as a result of aging, immunosuppressive medication use, endocrine disorders, malnourishment, extended use of medical equipment, and an increase in immunogenic diseases. Despite C. albicans being the species most frequently associated with human infections, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei also have been identified. Several antifungal drugs with different modes of action are approved for use in clinical settings to treat fungal infections. However, due to the common eukaryotic structure of humans and fungi, only a limited number of antifungal drugs are available for therapeutic use. Furthermore, drug resistance in Candida species has emerged as a result of the growing use of currently available antifungal drugs against fungal infections. Amphotericin B (AmB), a polyene class of antifungal drugs, is mainly used for the treatment of serious systemic fungal infections. AmB interacts with fungal plasma membrane ergosterol, triggering cellular ion leakage via pore formation, or extracting the ergosterol from the plasma membrane inducing cellular death. AmB resistance is primarily caused by changes in the content or structure of ergosterol. This review summarizes the antifungal drug resistance exhibited by Candida species, with a special focus on AmB.

Brain glucose induces tolerance of Cryptococcus neoformans to amphotericin B during meningitis.

Antibiotic tolerance is the ability of a susceptible population to survive high doses of cidal drugs and has been shown to compromise therapeutic outcomes in bacterial infections. In comparison, whether fungicide tolerance can be induced by host-derived factors during fungal diseases remains largely unknown. Here, through a systematic evaluation of metabolite-drug-fungal interactions in the leading fungal meningitis pathogen, Cryptococcus neoformans, we found that brain glucose induces fungal tolerance to amphotericin B (AmB) in mouse brain tissue and patient cerebrospinal fluid via the fungal glucose repression activator Mig1. Mig1-mediated tolerance limits treatment efficacy for cryptococcal meningitis in mice via inhibiting the synthesis of ergosterol, the target of AmB, and promoting the production of inositolphosphorylceramide, which competes with AmB for ergosterol. Furthermore, AmB combined with an inhibitor of fungal-specific inositolphosphorylceramide synthase, aureobasidin A, shows better efficacy against cryptococcal meningitis in mice than do clinically recommended therapies.

Comparative study on the anti-tumor effect of steroids derived from different organisms in H22 tumor-bearing mice and analysis of their mechanisms.

This study aimed to comparatively investigate the anti-tumor mechanisms of steroids including ergosterol, ß-sitosterol, cholesterol, and fucosterol. The model of H22 tumor-bearing mice was constructed based on histopathological data and biochemical parameters, while serums were subjected to metabolomics analysis to study the potential anti-tumor mechanisms. The results indicated that the four steroids exhibited different degrees of anti-tumor effects on H22 mice. The tumor inhibition rates were 63.25% for ergosterol, 56.41% for ß-sitosterol, 61.54% for cholesterol, and 72.65% for fucosterol. Metabolomic analyses revealed that 87, 71, and 129 differential metabolites were identified in ergosterol, cholesterol, and fucosterol treatment groups, respectively. The fucosterol treatment group had the highest number of differential metabolites. At the same time, it mainly inhibited purine and amino acid metabolism to exert anti-tumor effects. Ergosterol enhanced immunity and affected pyruvate metabolism, and cholesterol inhibited purine metabolism. The chemical structure difference among ergosterol, cholesterol, and fucosterol is mainly at the number and position of sterol double bonds and the number and length of side chain carbons. Therefore, there is a structure-activity relationship between the structure of steroid compounds and their efficacy. This study provides a key foundation for the exploitation of the anti-tumor effects of steroids derived from different organisms.

Emerging Role of Sphingolipids in Amphotericin B Drug Resistance.

Invasive fungal infections in humans are common in people with compromised immune systems and are difficult to treat, resulting in high mortality. Amphotericin B (AmB) is one of the main antifungal drugs available to treat these infections. AmB binds with plasma membrane ergosterol, causing leakage of cellular ions and promoting cell death. The increasing use of available antifungal drugs to combat pathogenic fungal infections has led to the development of drug resistance. AmB resistance is not very common and is usually caused by changes in the amount or type of ergosterol or changes in the cell wall. Intrinsic AmB resistance occurs in the absence of AmB exposure, whereas acquired AmB resistance can develop during treatment. However, clinical resistance arises due to treatment failure with AmB and depends on multiple factors such as the pharmacokinetics of AmB, infectious fungal species, and host immune status. Candida albicans is a common opportunistic pathogen that can cause superficial infections of the skin and mucosal surfaces, thrush, to life-threatening systemic or invasive infections. In addition, immunocompromised individuals are more susceptible to systemic infections caused by Candida, Aspergillus, and Cryptococcus. Several antifungal drugs with different modes of action are used to treat systemic to invasive fungal infections and are approved for clinical use in the treatment of fungal diseases. However, C. albicans can develop a variety of defenses against antifungal medications. In fungi, plasma membrane sphingolipid molecules could interact with ergosterol, which can lead to the alteration of drug susceptibilities such as AmB. In this review, we mainly summarize the role of sphingolipid molecules and their regulators in AmB resistance.

Retinoic acid shows direct parasiticidal activity by targeting ergosterol pathway in Leishmania donovani: a potential therapeutic advancement.

Visceral leishmaniasis (VL) is an infectious disease caused by Leishmania donovani parasite in Indian subcontinent and is life-threatening. It primarily inflicts the malnourished population. There is little therapeutic advancement in the last one decade or more, as the available drugs show adverse effects, complex long treatment, high cost and drug resistance. Here, in a concerted approach, we intended to address the malnutrition as well as the parasite load with a single modality. Our earlier findings show the protective effects of retinoic acid (RA) in controlling the parasite load in infected macrophages (mφ) and restores their M1 phenotype. RA also restores the levels of cellular cholesterol in infected mφ. In this process, we observed loss of ergosterol in the parasite upon treatment with RA. Hence, we hypothesized that RA, besides boosting the parasiticidal mechanism in mφ, may also target the sterol pathway in the parasite by targeting sterol 24-C methyltransferase (SMT). SMT plays an essential role in the formation of ergosterol, required for growth and viability in Leishmania species. Therefore, we predicted as well as validated the 3D structure of SMT protein and performed the quality check. RA showed -9.9 free binding energy towards SMT which is higher than any of its derivatives. The molecular dynamics showed stable conjugate and the in vitro testing showed a reduction by ∼ twofold in the parasite number upon RA treatment. Importantly, it showed a loss of ergosterol possibly due to the inhibition of SMT protein. Our finding showed direct parasiticidal function of RA which is of significance in terms of therapeutic advancement.Communicated by Ramaswamy H. Sarma.

Alterations in the Level of Ergosterol in Candida albicans' Plasma Membrane Correspond with Changes in Virulence and Result in Triggering Diversed Inflammatory Response.

Opportunistic pathogen Candida albicans possesses multiple virulence factors which enable colonization and infection of host tissues. Candida-related infections frequently occur in immunocompromised patients, which is related to an insufficient inflammatory response. Furthermore, immunosuppression and multidrug resistance of C. albicans clinical isolates make the treatment of candidiasis a challenge for modern medicine. The most common resistance mechanism of C. albicans to antifungals includes point mutations in the ERG11 gene, which encodes target protein for azoles. We investigated whether the mutations or deletion of the ERG11 gene influence the pathogen-host interactions. We prove that both C. albicans erg11∆/∆ and ERG11K143R/K143R exhibit increased cell surface hydrophobicity. Additionally, C. albicans KS058 has an impaired ability of biofilm and hyphae formation. Analysis of the inflammatory response of human dermal fibroblasts and vaginal epithelial cell lines revealed that altered morphology of C. albicans erg11∆/∆ results in a significantly weaker immune response. C. albicans ERG11K143R/K143R triggered stronger production of pro-inflammatory response. Analysis of genes encoding adhesins confirmed differences in the expression pattern of key adhesins for both erg11∆/∆ and ERG11K143R/K143R strains. Obtained data indicate that alterations in Erg11p consequence in resistance to azoles and affect the key virulence factors and inflammatory response of host cells.

A Novel Study of Correlation of Lipid Parameters with Clinical Profile, Staging and Onset of Rhino Orbito Cerebral Mucormycosis Covid 19 Pandemic.

Mucormycosis is an angioinvasive disease caused by mold fungi of the genus Rhizopus, Mucor. India has reported surge in cases of COVID 19 associated Mucormycosis over the past few months due to the increasing frequency of risk factors like corticosteroid therapy, uncontrolled diabetes, neutropenia and obesity. Studies have shown that eukaryote cell membrane contains cholesterol and fungal cell wall contains ergosterol with lanosterol being precursor for both and ergosterol is essential for mitochondrial DNA maintenance in fungi, as cholesterol is in humans. The current study is based on the hypothesis that fungi can use human cholesterol as a raw material to maintain its cell function and accentuate its own multiplication and this can indirectly be shown by the association between deranged lipid parameters in an individual with severity of Mucormycosis. Thus present study aims to estimate the lipid parameters and correlate the serum lipid parameters with clinical profile, stage of the disease and duration of onset of mucormycosis in patients with COVID associated Mucormycosis. MATERIAL: This is a cross sectional study conducted on 103 patients diagnosed with COVID 19 associated mucormycosis admitted to the hospitals attached to BMCRI from July 2021 to September 2021. Serum fasting lipid profile and other biochemical parameters were determined. The correlation of lipid levels with clinical profile, onset and staging of mucormycosis patients were obtained. OBSERVATION: The age distribution varied from 22yrs to 75yrs of whom majority were males (83.4%). Among patients with mucormycosis of all severity stages, nasal block (79.6%) was found to be most common symptom followed by headache(75.7%). Among patients with mucormycosis most frequent associated comorbidity was Diabetes mellitus (DM) followed by hypertension (HTN) followed by DM and HTN followed by Ischemic Heart Disease (IHD) followed by DM,HTN and IHD. The study showed statistically significant correlation such that severity of mucormycosis increased with progressive worsening lipid parameters. It also showed statistically significant correlation such that patients with increasing TC,LDL,VLDL,TG levels had shorter COVID 19 onset to mucormycosis onset duration. CONCLUSION: The study showed a positive correlation between serum lipid profile and staging of mucormycosis and negative correlation between lipid levels with duration between onset of COVID 19 to onset of mucormycosis. Hence serum lipid profile can be used as an excellent marker to predict the severity and prognosis of COVID 19 associated mucormycosis.

Antiviral effects of ergosterol peroxide in a pig model of porcine deltacoronavirus (PDCoV) infection involves modulation of apoptosis and tight junction in the small intestine.

Porcine deltacoronavirus (PDCoV) is a newly discovered swine enteropathogenic coronavirus with worldwide distribution. However, efficient strategies to prevent or treat the infection remain elusive. Our in vitro study revealed that ergosterol peroxide (EP) from the mushroom Cryptoporus volvatus has efficient anti-PDCoV properties. The aim of this study is to evaluate the potential of EP as a treatment for PDCoV in vivo and elucidate the possible mechanisms. Seven-day-old piglets were infected with PDCoV by oral administration in the presence or absence of EP. Piglets infected with PDCoV were most affected, whereas administration of EP reduced diarrhea incidence, alleviated intestinal lesion, and decreased viral load in feces and tissues. EP reduced PDCoV-induced apoptosis and enhanced tight junction protein expressions in the small intestine, maintaining the integrity of the intestinal barrier. EP showed immunomodulatory effect by suppressing PDCoV-induced pro-inflammatory cytokines and the activation of IκBα and NF-κB p65, and upregulating IFN-I expression. Knockdown of p38 inhibited PDCoV replication and alleviated PDCoV-induced apoptosis, implying that EP inhibited PDCoV replication and alleviated PDCoV-induced apoptosis via p38/MAPK signaling pathway. Collectively, ergosterol peroxide can protect piglets from PDCoV, revealing the potential of EP for development as a promising strategy for treating and controlling the infection of PDCoV.

Separation and identification of tubocapsanolide MAP and tubocapsunolide A, and the structure-activity relationship of their anti-TNBC activity.

A new withanolide, tubocaapsanolide MAP (MeOH addition product) (1), as well as its known precursor tubocaapsanolide A (2) were obtained from Tubocapsicum anomalum (Solanaceae). Compound 1 was a MeOH addition product transformed from compound 2 during the process of separation using MeOH as solvent. The structures of the two withanolides including absolute configuration were elucidated by extensive spectroscopic analysis and X-ray single crystal diffraction. In the test of anti-triple negative breast cancer (TNBC) effects, tubocapsusanlide A (2) showed potent inhibitory activity against four human TNBC cell lines, while tubocapsusanlide MAP (1) exhited significantly weaker inhibitory than that of tubocapsusanlide A (2), indicating that α-ß unsaturated carbonyl unit contained in 2 was closely related to its anti-TNBC activity. The potent bioactivity displayed significant developing potential of withanolides as anti-TNBC lead compounds or drug candidates. And this report may provide some useful guidances for the preparation and bioactivity research of withanolides.

Examining the health effects and bioactive components in Agaricus bisporus mushrooms: a scoping review.

There is evidence from both in vitro and animal models that the consumption of edible mushrooms has beneficial effects on health. It is unclear whether similar effects exist in humans and which bioactive compounds are present. This review synthesises the evidence on the world's most commonly consumed mushroom, Agaricus bisporus to (i) examine its effect on human health outcomes; and (ii) determine the nutrient density of its bioactive compounds, which may explain their health effects. A systematic literature search was conducted on the consumption of A. bisporus, without date and study design limits. Bioactive compounds included ergosterol, ergothioneine, flavonoids, glucans and chitin. Two authors independently identified studies for inclusion and assessed methodological quality. Beneficial effects of A. bisporus on metabolic syndrome, immune function, gastrointestinal health and cancer, with the strongest evidence for the improvement in Vitamin D status in humans, were found. Ultraviolet B (UVB) exposed mushrooms may increase and maintain serum 25(OH)D levels to a similar degree as vitamin D supplements. A. bisporus contain beta-glucans, ergosterol, ergothioneine, vitamin D and an antioxidant compound usually reported as flavonoids; with varying concentrations depending on the type of mushroom, cooking method and duration, and UVB exposure. Further research is required to fully elucidate the bioactive compounds in mushrooms using vigorous analytical methods and expand the immunological markers being tested. To enable findings to be adopted into clinical practice and public health initiatives, replication of existing studies in different population groups is required to confirm the impact of A. bisporus on human health.

Inhibitory effect of ergosterol on bladder carcinogenesis is due to androgen signaling inhibition by brassicasterol, a metabolite of ergosterol.

We previously revealed that Choreito, a traditional Kampo medicine, strongly inhibits bladder carcinogenesis promotion. We have also shown that Polyporus sclerotium, which is one of the crude drugs in Choreito, has the strongest bladder carcinogenesis inhibitory effect and that the ergosterol contained in Polyporus sclerotium is the main active component. In this study, we analyzed the mechanism by which ergosterol inhibits bladder carcinogenesis. Rats were given an N-butyl-N-(4-hydroxybutyl) nitrosamine (BHBN) solution ad libitum, and then a promoter [saccharin sodium (SS), DL-tryptophan, or BHBN] was administered together with ergosterol or its metabolite, brassicasterol. The bladders were removed from rats, and the inhibitory effect on carcinogenesis promotion was evaluated by an agglutination assay with concanavalin A (Con A). Although the oral administration of ergosterol inhibited the promotion of bladder carcinogenesis with SS, the intraperitoneal administration of brassicasterol showed a stronger effect. The effect of brassicasterol on carcinogenesis promotion was observed regardless of the type of promoter. Administration of testosterone to castrated rats increased the number of cell aggregates caused by Con A. In contrast, intraperitoneal administration of brassicasterol to castrated rats treated with testosterone significantly decreased the number of cell aggregates, confirming the inhibition of bladder carcinogenesis promotion. The inhibitory effect of ergosterol on bladder carcinogenesis is due to brassicasterol, a metabolite of ergosterol. The action of brassicasterol via androgen signaling may play a role in the inhibitory effect on bladder carcinogenesis promotion.

Optimization of ergosterol extraction from Pleurotus mushrooms using response surface methodology.

In this study, heat-assisted extraction (HAE) was used to maximize ergosterol extraction from Pleurotus ostreatus (PO) and Pleurotus eryngii (PE) using response surface methodology (RSM). Different temperature (T) and time (t) conditions were applied to understand their influence on the extraction yield (Y1), ergosterol purity in the extracted material (mg g-1 R, Y2) and ergosterol content in the two Pleurotus species (mg per 100 g dw, Y3). A circumscribed central composite design was used to evaluate the interactive effects of extraction variables and the optimal conditions were determined using second-order polynomial mathematical models to describe the responses obtained. In all cases, the predicted responses showed satisfactory fitting between the predicted and experimental values (R2 values >0.8). The global optimum conditions predicted by the models were for PO at T = 54.3 °C and t = 150 min (yielded 7.25%, 33.32 mg E per g R and 244.25 mg E per 100 g dw), while for PE at T = 61.8 °C and t = 150 min (yielded 8.02%, 43.63 mg E per g R and 360.58 mg E per 100 g dw). The obtained results from the two Pleurotus mushroom species using HAE show the possibilities of using them as a production source of enriched extracts in ergosterol.

Ergosterol Peroxide from the Medicinal Mushroom Ganoderma lucidum Inhibits Differentiation and Lipid Accumulation of 3T3-L1 Adipocytes.

Ergosterol peroxide is a natural compound of the steroid family found in many fungi, and it possesses antioxidant, anti-inflammatory, anticancer and antiviral activities. The anti-obesity activity of several edible and medicinal mushrooms has been reported, but the effect of mushroom-derived ergosterol peroxide on obesity has not been studied. Therefore, we analyzed the effect of ergosterol peroxide on the inhibition of triglyceride synthesis at protein and mRNA levels and differentiation of 3T3-L1 adipocytes. Ergosterol peroxide inhibited lipid droplet synthesis of differentiated 3T3-L1 cells, expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAT/enhancer-binding protein alpha (C/EBPα), the major transcription factors of differentiation, and also the expression of sterol regulatory element-binding protein-1c (SREBP-1c), which promotes the activity of PPARγ, resulting in inhibition of differentiation. It further inhibited the expression of fatty acid synthase (FAS), fatty acid translocase (FAT), and acetyl-coenzyme A carboxylase (ACC), which are lipogenic factors. In addition, it inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) involved in cell proliferation and activation of early differentiation transcription factors in the mitotic clonal expansion (MCE) stage. As a result, ergosterol peroxide significantly inhibited the synthesis of triglycerides and differentiation of 3T3-L1 cells, and is, therefore, a possibile prophylactic and therapeutic agent for obesity and related metabolic diseases.

The key role of macrophage depolarization in the treatment of COPD with ergosterol both in vitro and in vivo.

Macrophages are the most abundant immune cells in the lung, which play an important role in COPD. The anti-inflammatory and anti-oxidation of ergosterol are well documented. However, the effect of ergosterol on macrophage polarization has not been studied. The objective of this work was to investigate the effect of ergosterol on macrophage polarization in CSE-induced RAW264.7 cells and Sprague-Dawley (SD) rats COPD model. Our results demonstrate that CSE-induced macrophages tend to the M1 polarization via increasing ROS, IL-6 and TNF-α, as well as increasing MMP-9 to destroy the lung construction in both RAW264.7 cells and SD rats. However, treatment of RAW264.7 cells and SD rats with ergosterol inhibited CSE-induced inflammatory by decreasing ROS, IL-6 and TNF-α, and increasing IL-10 and TGF-ß, shuffling the dynamic polarization of macrophages from M1 to M2 both in vitro and in vivo. Ergosterol also decreased the expression of M1 marker CD40, while increased that of M2 marker CD163. Moreover, ergosterol improved the lung characters in rats by decreasing MMP-9. Furthermore, ergosterol elevated HDAC3 activation and suppressed P300/CBP and PCAF activation as well as acetyl NF-κB/p65 and IKKß, demonstrating that HDAC3 deacetylation was involved in the effect of ergosterol on macrophage polarization. These results also provide a proof in immunoregulation of ergosterol for therapeutic effects of cultured C. sinensis on COPD patients.

Ergosteryl 2-naphthoate, An Ergosterol Derivative, Exhibits Antidepressant Effects Mediated by the Modification of GABAergic and Glutamatergic Systems.

Phytosterols are a kind of natural component including sitosterol, campesterol, avenasterol, ergosterol (Er) and others. Their main natural sources are vegetable oils and their processed products, followed by grains, by-products of cereals and nuts, and small amounts of fruits, vegetables and mushrooms. In this study, three new Er monoester derivatives were obtained from the reflux reaction with Er: organic acids (furoic acid, salicylic acid and 2-naphthoic acid), 1-Ethylethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) in dichloromethane. Their chemical structures were defined by IR and NMR. The present study was also undertaken to investigate the antidepressant-like effects of Er and its derivatives in male adult mice models of depression, and their probable involvement of GABAergic and glutamatergic systems by the forced swim test (FST). The results indicated that Er and its derivatives display antidepressant effects. Moreover, one derivative of Er, ergosteryl 2-naphthoate (ErN), exhibited stronger antidepressant activity in vivo compared to Er. Acute administration of ErN (5 mg/kg, i.p.) and a combination of ErN (0.5 mg/kg, i.p.), reboxetine (2.5 mg/kg, i.p.), and tianeptine (15 mg/kg, i.p.) reduced the immobility time in the FST. Pretreatment with bicuculline (a competitive γ-aminobutyric acid (GABA) antagonist, 4 mg/kg, i.p.) and N-methyl-d-aspartic acid (NMDA, an agonist at the glutamate site, 75 mg/kg, i.p.) effectively reversed the antidepressant-like effect of ErN (5 mg/kg, i.p.). However, prazosin (a α1-adrenoceptor antagonist, 1 mg/kg, i.p.) and haloperidol (a non-selective D2 receptor antagonist, 0.2 mg/kg, i.p.) did not eliminate the reduced immobility time. Altogether, these results indicated that ErN produced antidepressant-like activity, which might be mediated by GABAergic and glutamatergic systems.

Effects of Ergosterol on COPD in Mice via JAK3/STAT3/NF-κB Pathway.

The present study was to evaluate the effect of ergosterol (ER) on CS (cigarette smoke)-induced chronic obstructive pulmonary disease (COPD) in mice. Fifty male ICR mice were randomly assigned to five groups: control group, CS group, CS + dexamethasone (Dex, 2 mg/kg) group, CS + ER (ER, 25 mg/kg) group, CS + ER (ER, 50 mg/kg). H&E staining demonstrated that ER inhibited CS-induced pathological injury in lung tissue. Besides, ER could restore the activities of superoxide dismutase (SOD) in serum and in the lung, catalase (CAT) in serum and reduce the content of malondialdehyde (MDA) in serum and in the lung. ER also inhibited pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) in serum and the lung. Furthermore, ER significantly inhibited the protein expression of JAK3/STAT3/NF-κB pathway in CS-induced mice. Our findings suggested that ER might effectively ameliorate the progression of COPD via JAK3/STAT3/NF-κB pathway in mice.

Cytotoxic effects of ergone, a compound isolated from Fulviformes fastuosus.

BACKGROUND: Mushrooms inspired the cuisines of many cultures and conventional medicaments for cancer. However, a substantial number of mushroom species are yet unexplored, possessing an unknown chemical, biological and pharmacological profiles. Fulviformes fastuosus is a terrestrial mushroom, which is commonly found in Sri Lankan woodlands. The current study was aimed at isolation and characterization of a potent cytotoxic compound from F. fastuosus and investigating the apoptotic effect induced by the active principle against cancer and normal cell lines. METHODS: Bioactivity guided isolation of active principles from the methanol extract of F. fastuosus was performed by a rapid extraction and isolation method using different chromatographic techniques. Potential cytotoxic compound was identified using one and two dimensional nuclear magnetic resonance spectroscopy and mass spectrometry. Isolated compound was screened for in vitro cytotoxicity against Hepatocellular carcinoma (HepG-2), Muscle rhabdomyosarcoma (RD) and Rat Wistar liver normal (CC-1) cell lines using 3 4, 5-(dimethylthiazol-2-yl) 2-5-diphenyl tetrazolium bromide (MTT) cell viability assay. Apoptotic features of cells were observed via microscopic examination and ethidium bromide/acridine orange fluorescent staining. RESULTS: The interpretation of spectral data resulted in the identification of the chemical structure as ergosta-4,6,8 (14),22-tetraen-3-one (ergone). Ergone exhibited promising cytotoxic properties against RD cells with less cytotoxicity effect on CC-1 cells. In addition, ergone also possesses a strong cytotoxic effect against HepG-2 cells showing low toxic level for CC-1 cells. Apoptotic features of treated cells were detected via morphological characterization and ethidium bromide/acridine orange staining. CONCLUSION: The present study elaborates the isolation of a potent cytotoxic compound; ergone, from F. fastuosus via a rapid and efficient isolation method. Importantly, ergone has exhibited greater cytotoxic activity against RD cells with high selectivity index compared to cytotoxicity against HepG-2 cells. Ergone can be used in the development of therapeutic strategies for curbing rhabdomyosarcoma.

Ergosterol peroxide from Chaga mushroom (Inonotus obliquus) exhibits anti-cancer activity by down-regulation of the ß-catenin pathway in colorectal cancer.

AIM OF THE STUDY: In this study, we examined the effect of different fractions and components of Chaga mushroom (Inonotus Obliquus) on viability and apoptosis of colon cancer cells. Among them, one component showed the most effective growth inhibition and was identified as ergosterol peroxide by NMR analysis. We investigated the anti-proliferative and apoptosis mechanisms of ergosterol peroxide associated with its anti-cancer activities in human colorectal cancer (CRC) cell lines and tested its anti-tumor effect on colitis-induced CRC developed by Azoxymethane (AOM)/Dextran sulfate sodium (DSS) in a mouse model. MATERIALS AND METHODS: We used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, flow cytometry assays, Western blot analysis, colony formation assays, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and AOM/DSS mouse models to study the molecular mechanism of metastatic activities in CRC cells. RESULTS: Ergosterol peroxide inhibited cell proliferation and also suppressed clonogenic colony formation in HCT116, HT-29, SW620 and DLD-1 CRC cell lines. The growth inhibition observed in these CRC cell lines was the result of apoptosis, which was confirmed by FACS analysis and Western blotting. Ergosterol peroxide inhibited the nuclear levels of ß-catenin, which ultimately resulted in reduced transcription of c-Myc, cyclin D1, and CDK-8. Ergosterol peroxide administration showed a tendency to suppress tumor growth in the colon of AOM/DSS-treated mice, and quantification of the IHC staining showed a dramatic decrease in the Ki67-positive staining and an increase in the TUNEL staining of colonic epithelial cells in AOM/DSS-treated mice by ergosterol peroxide for both prevention and therapy. CONCLUSION: Our data suggest that ergosterol peroxide suppresses the proliferation of CRC cell lines and effectively inhibits colitis-associated colon cancer in AOM/DSS-treated mice. Ergosterol peroxide down-regulated ß-catenin signaling, which exerted anti-proliferative and pro-apoptotic activities in CRC cells. These properties of ergosterol peroxide advocate its use as a supplement in colon cancer chemoprevention.

Beneficial effects of the traditional medicine Igongsan and its constituent ergosterol on dextran sulfate sodium-induced colitis in mice.

Ulcerative colitis (UC) is a type of inflammatory bowel disease and is considered a chronic gastrointestinal disorder. Igongsan (IGS) is a Korean herbal medicine, which has been used to treat digestive disorders. However, the ameliorative effect and molecular mechanisms of IGS in intestinal inflammation have not yet been studied in detail. The present study aimed to investigate the protective effects of IGS and its constituent, ergosterol, in a mouse model of dextran sulfate sodium (DSS)­induced colitis. Colitis was induced in mice by supplementing their drinking water with 5% (w/v) DSS for 7 days. The effects of IGS were then determined on DSS­induced clinical signs of colitis, including weight loss, colon shortening, diarrhea and obscure/gross bleeding. In addition, the effects of IGS were determined on the expression levels of inflammation­associated genes in the colon tissue of DSS­treated mice. The results of the present study demonstrated that mice treated with DSS exhibited marked clinical symptoms, including weight loss and reduced colon length. Treatment with IGS attenuated these symptoms and also suppressed the expression levels of tumor necrosis factor­α and interleukin­6, as well as the expression of cyclooxygenase­2 in the colon tissue of DSS­treated mice. IGS also reduced the activation of the transcription factor nuclear factor­κB p65 in the colon tissue of DSS­treated mice. In addition, ergosterol was shown to attenuate the DSS­induced clinical symptoms of colitis in mice. In conclusion, the present study provided experimental evidence that IGS may be a useful therapeutic drug for patients with UC.

Effects of Ergosterol, Isolated from Scleroderma Polyrhizum Pers., on Lipopolysaccharide-Induced Inflammatory Responses in Acute Lung Injury.

The present study aimed to investigate the protective role of ergosterol, isolated from Scleroderma polyrhizum Pers., in lipopolysaccharide (LPS)-induced acute lung injury (ALI). ALI was induced in mice by LPS (0.5 mg/kg), and ergosterol (25 and 50 mg/kg) was administrated orally 1 h prior to LPS administration. Ergosterol pretreatment at doses of 25 and 50 mg/kg decreased LPS-induced lung histopathological changes, lung wet-to-dry weight ratio. In addition, pretreatment with ergosterol inhibited inflammatory cells and proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Furthermore, we demonstrated that ergosterol blocked the activation of nuclear factor-kappaB (NF-κB), cyclooxygenase (COX)-2, and nitric oxide synthase (iNOS) pathways. The results presented here suggest that the protective mechanism of ergosterol may be attributed partly to the inhibition of NF-κB, COX-2, and iNOS pathways.