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.

Synthesis, Antibacterial Activity and Molecular Docking of Phospholidinones in Stigmastane Series.

INTRODUCTION: Steroid compounds are widely distributed in nature throughout scientific history. Living organisms such as animals and vegetables have steroids that show a significant effect on their vital activities. Sterols are key components of all eukaryotic cell membranes. METHODS: Steroidal compounds; 3ß-oxo-[1',3',2'-oxathiaphos-phalidine-2'-one] stigmast-5-ene and 3ß- oxo[1`,3`,2`-dioxaphosphalidine-2`-one]-stigmast-5-ene were successfully prepared using easily accessible 3ß-hydroxy stigmast-5-ene with phosphorous oxychloride (POCl3), 2- mercaptoethanol/ethylene glycol and triethylamine (Et3N) in dry diethyl ether. Products were obtained in semi-solid state and characterized using physicochemical techniques. RESULTS: The results of the bioassay showed that the synthesized compound containing the sulfur atom had antibacterial activity. Molecular docking was also done in order to show in silico antibacterial activity and to make out the probable binding mode of compound with the amino acid residues of protein. CONCLUSION: The results of the docking study showed that synthesized compound 2 had minimal binding energy with substantial affinity for the active site.

Neo-5,22E-Cholestadienol Derivatives from Buthus martensii Karsch and Targeted Bactericidal Action Mechanisms.

The discovery and search for new antimicrobial molecules from insects and animals that live in polluted environments is a very important step in the scientific search for solutions to the current problem of antibiotic resistance. Previously, we have reported that the secondary metabolite with the antibacterial action discovered in scorpion. The current study further isolated three new compounds from Buthus martensii karsch, while compounds 1 and 2 possessed 5,22E-cholestadienol derivatives whose structure demonstrated broad spectrum bactericide activities. To explore the antibacterial properties of these new compounds, the result shows that compound 2 inhibited bacterial growth of both S. aureus and P. aeruginosa in a bactericidal rather than a bacteriostatic manner (MBC/MIC ratio ≤ 2). Similarly, with compound 1, a ratio of MBC/MIC ≤ 2 indicates bactericidal activity inhibited bacterial growth of P. aeruginosa. Remarkably, this suggests that two compounds can be classified as bactericidal agents against broad spectrum bactericide activities for 5,22E-cholestadienol derivatives from Buthus martensii karsch. The structures of compounds 1⁻3 were established by comprehensive spectra analysis including two-dimensional nuclear magnetic resonance (2D-NMR) and high-resolution electrospray ionization-mass spectrometry (HRESI-MS) spectra. The antibacterial mechanism is the specific binding (various of bonding forces between molecules) using compound 1 or 2 as a ligand based on the different receptor proteins'-2XRL or 1Q23-active sites from bacterial ribosome unit A, and thus prevent the synthesis of bacterial proteins. This unique mechanism avoids the cross-resistance issues of other antibacterial drugs.

Fucosterol Causes Small Changes in Lipid Storage and Brassicasterol Affects some Markers of Lipid Metabolism in Atlantic Salmon Hepatocytes.

Several feeding trials with Atlantic salmon fed naturally high phytosterol concentrations due to dietary rapeseed oil inclusion have shown changes in lipid metabolism and increased hepatic lipid storage in the fish. An in vitro trial with Atlantic salmon hepatocytes was, therefore, performed to study the possible direct effects of phytosterols on lipid storage and metabolism. The isolated hepatocytes were exposed to seven different sterol treatments and gene expression, as well as lipid accumulation by Oil Red O dyeing, was assessed. Fucosterol, a sterol found in many algae species, had an effect on the size of individual lipid droplets, leading to smaller lipid droplets than in the control without added sterols. A sterol extract from soybean/rapeseed led to an increase in the percentage of hepatocytes with visible lipid droplets at 20× magnification, while hepatocytes of both the sterol extract-treated groups and fucosterol-treated groups had a larger proportion of their area covered with lipids compared to control cells. Brassicasterol, a sterol characteristic of rapeseed oil, was the only sterol treatment leading to a change in gene expression, affecting the expression of the nuclear receptors, peroxisome proliferator-activated receptor gamma (pparg) and retinoid X receptor (rxr). The current study thus shows that phytosterols can have direct, although subtle, effects on both hepatic lipid storage and gene expression of Atlantic salmon in vitro.

Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design.

Ergosterol biosynthesis pathways essential to pathogenic protozoa growth and absent from the human host offer new chokepoint targets. Here, we present characterization and cell-based interference of Acanthamoeba spp sterol 24-/28-methylases (SMTs) that catalyze the committed step in C28- and C29-sterol synthesis. Intriguingly, our kinetic analyses suggest that 24-SMT prefers plant cycloartenol whereas 28-SMT prefers 24(28)-methylene lophenol in similar fashion to the substrate preferences of land plant SMT1 and SMT2. Transition state analog-24(R,S),25-epiminolanosterol (EL) and suicide substrate 26,27-dehydrolanosterol (DHL) differentially inhibited trophozoite growth with IC50 values of 7 nM and 6 µM, respectively, and EL yielded 20-fold higher activity than reference drug voriconazole. Against either SMT assayed with native substrate, EL exhibited tight binding ∼Ki 9 nM. Alternatively, DHL is methylated at C26 by 24-SMT that thereby, generates intermediates that complex and inactivate the enzyme, whereas DHL is not productively bound to 28-SMT. Steroidal inhibitors had no effect on human epithelial kidney cell growth or cholesterol biosynthesis at minimum amoebicidal concentrations. We hypothesize the selective inhibition of Acanthamoeba by steroidal inhibitors representing distinct chemotypes may be an efficient strategy for the development of promising compounds to combat amoeba diseases.

Insights into structure and activity of natural compound inhibitors of pneumolysin.

Pneumolysin is the one of the major virulence factor of the bacterium Streptococcus pneumoniae. In previous report, it is shown that ß-sitosterol, a natural compound without antimicrobial activity, is a potent antagonist of pneumolysin. Here, two new pneumolysin natural compound inhibitors, with differential activity, were discovered via haemolysis assay. To explore the key factor of the conformation for the inhibition activity, the interactions between five natural compound inhibitors with differential activity and pneumolysin were reported using molecular modelling, the potential of mean force profiles. Interestingly, it is found that incorporation of the single bond (C22-C23-C24-C25) to replace the double bond (hydrocarbon sidechain) improved the anti-haemolytic activity. In view of the molecular modelling, binding of the five inhibitors to the conserved loop region (Val372, Leu460, and Tyr461) of the cholesterol binding sites led to stable complex systems, which was consistent with the result of ß-sitosterol. Owing to the single bond (C22-C23-C24-C25), campesterol and brassicasterol could form strong interactions with Val372 and show higher anti-haemolytic activity, which indicated that the single bond (C22-C23-C24-C25) in inhibitors was required for the anti-haemolytic activity. Overall, the current molecular modelling work provides a starting point for the development of rational design and higher activity pneumolysin inhibitors.

New Bioactive Semisynthetic Derivatives of 31-Norlanostenol and Obtusifoliol from Euphorbia officinarum.

Fifteen semisynthetic terpenoid derivatives from the major latex components of Euphorbia officinarum have been evaluated against the insect pest Spodoptera littoralis, two species of protozoan parasites, Trypanosoma cruzi and Leishmania infantum, and also against insect Sf9 and mammalian CHO cells to test their selective cytotoxicity. Our results showed that 40% of the test substances were postingestive toxicants to S. littoralis. All the tested derivatives had cytotoxic effects on insect-derived Sf9 cells, whereas mammalian CHO cells were affected by a lower number of compounds (47%). Furthermore, 87% of the test compounds had antiparasitic effects on both L. infantum and T. cruzi, with some of them being selective parasite toxicants.

Competitive Solubilization of Cholesterol/Cholesteryl Oleate and Seven Species of Sterol/Stanol in Model Intestinal Solution System.

The addition of plant sterols/stanols (sterols or stanols) can reduce the solubilization of cholesterol in a model intestinal solution system. We studied the molecular structure of seven different sterols/stanols and the effect they had on the solubilization of cholesterol or cholesterol ester in a model intestinal solution. The differences in the molecular structures of the sterol/stanol species influenced their abilities to reduce the solubility of cholesterol in the competitive solubilization experiments. Cholestanol whose molecular structure resembled cholesterol was the most effective at reducing the solubilization of cholesterol and cholesterol ester, with the solubilities of cholesterol and cholesteryl oleate being 41% and 39% respectively of the values observed for the single solubilizate systems. ß-Sitosterol was also able to reduce the solubilities of cholesterol and cholesteryl oleate to 43% and 45% of those observed in a single solubilizate system. Both, stigmasterol and brassicasterol have an unsaturated double bond in a steroid side chain and did not exhibit major cholesterol-lowering effects. These results were reflected by the Gibbs free energy change values (ΔG(0)) for solubilization, where the sterol/stanol species with cholesterol-lowering effects had similar or larger negative ΔG(0) values than those observed for cholesterol.

Effects of margarine enriched with plant sterol esters from rapeseed and tall oils on markers of endothelial function, inflammation and hemostasis.

BACKGROUND AND AIMS: The sterol profile of rapeseed oil differs from that of tall oil with higher contents of campesterol and brassicasterol. We previously found that margarines providing 2 g/day of sterols from rapeseed or tall oil resulted in similar reductions in LDL cholesterol of 8-9%. The aim of the present study was to investigate whether the consumption of these margarines affected markers of endothelial function, inflammation and hemostasis. METHODS: Blood samples were collected from 58 hypercholesterolemic volunteers who completed a double-blinded, randomized, crossover trial. Subjects consumed each of the two sterol margarines and a control non-sterol margarine for 4 weeks separated by one-week washout periods. All the margarines had the same fatty acid composition. Concentrations of vascular cell adhesion molecule-l (VCAM-1), E-selection, circulating tumor necrosis factor α (TNFα) and plasminogen activator inhibitor-1 (total, tPAI-1; active, PAI-1) were quantified. RESULTS: Rapeseed-sterol margarine reduced E-selection concentrations compared to the control margarine (p = 0.012) while tall-sterol margarine had no effect. The rapeseed-sterol margarine also reduced tPAI-1 (p = 0.008) compared to the tall-sterol margarine. No significant changes were observed in TNFα and VCAM-1. No association was found between LDL reduction and changes in E-selection and tPAI-1. CONCLUSION: Rapeseed-sterol margarine demonstrated favorable effects on vascular risk markers.

Cytotoxic 5α,8α-epidioxy sterols from the marine sponge Monanchora sp.

Three new sterols, 5α,8α-epidioxy-24-norcholesta-6,9(11),22-trien-3ß-ol (1), 5α,8α-epidioxy-cholesta-6,9(11),24-trien-3ß-ol (2), and 5α,8α-epidioxy-cholesta-6,23-dien-3ß,25-diol (3), with four known sterols (4-7) were isolated from a marine sponge Monanchora sp. Their chemical structures were elucidated by extensive spectroscopic analysis. Compounds 1 and 3-7 showed moderate cytotoxicity against several human carcinoma cell lines including renal (A-498), pancreatic (PANC-1 and MIA PaCa-2), and colorectal (HCT 116) cancer cell lines.

New secondary metabolites from bioactive extracts of the fungus Armillaria tabescens.

Ethyl acetate extracts of Armillaria tabescens (strain JNB-OZ344) showed significant fungistatic and bacteristatic activities against several major human pathogens including Candida albicans, Cryptococcus neoformans, Escherichia coli and Mycobacterium intracellulare. Chemical analysis of these extracts led to the isolation and identification of four new compounds, emestrin-F (1), emestrin-G (2), 6-O-(4-O-methyl-ß-D-glucopyranosyl)-8-hydroxy-2,7-dimethyl-4H-benzopyran-4-one (3) and cephalosporolide-J (4), along with five other previously known compounds, emestrin (5), cephalosporolide-E (6), decarestrictine-C2 (7), ergosterol and brassicasterol. Structural elucidation of all compounds was carried out by NMR and MS analyses. Antimicrobial assays revealed that compounds 1 and 5 were responsible for the observed growth inhibitory activities of the fungal extracts against the human pathogens tested.

Soyasapogenol B and trans-22-dehydrocam- pesterol from common vetch (Vicia sativa L.) root exudates stimulate broomrape seed germination.

BACKGROUND: Orobanche and Phelipanche species (the broomrapes) are root parasitic plants, some of which represent serious weed problems causing severe yield losses on important crops. Control strategies have largely focused on agronomic practices, resistant crop varieties and herbicides, albeit with marginal success. An alternative control method is the induction of suicidal seed germination with natural substances isolated from root exudates of host and non-host plants. RESULTS: Soyasapogenol B [olean-12-ene-3,22,24-triol(3ß,4ß,22ß)] and trans-22-dehydrocampesterol [(ergosta-5,22-dien-3-ol, (3ß,22E,24S)] were isolated from Vicia sativa root exudates. They were identified by comparing their spectroscopic and optical properties with those reported in the literature. Soyasapogenol B was very specific, stimulating the germination of O. minor seeds only, whereas trans-22-dehydrocampesterol stimulated P. aegyptiaca, O. crenata, O. foetida and O. minor. CONCLUSION: Soyasapogenol B and trans-22-deydrocampesterol were isolated for the first time from Vicia sativa root exudates, and their biological activity as stimulants of Orobanche or Phelipanche sp. seed germination was reported.

Dietary phytosterols and phytostanols alter the expression of sterol-regulatory genes in SHRSP and WKY inbred rats.

BACKGROUND/AIMS: We elucidated the molecular mechanism(s) underlying sterol trafficking by investigating alterations in gene expression in response to increased retention of dietary phytosterols and phytostanols in stroke-prone spontaneously hypertensive (SHRSP) and normotensive Wistar Kyoto (WKY) inbred rats. METHODS: SHRSP and WKY inbred rats were fed a control diet or a diet supplemented with phytosterols or phytostanols (2 g/kg diet). RESULTS: Intake of phytosterols and phytostanols increased their incorporation in plasma, red blood cells, liver, aorta and kidney, but decreased cholesterol levels in liver and aorta in both rat strains. Phytosterol intake up-regulated mRNA expression of intestinal Npc1l1 and Abcg8, and hepatic Abcg5, Abca1, Cyp27a1 and Hmgcr. Phytostanol intake up-regulated Npc1l1 and Srebp2, but down-regulated Abcg5 mRNA expression in small intestine. Phytostanols also up-regulated Abca1 expression in SHRSP rats, but down-regulated Abca1 expression in WKY inbred rats. Compared to phytosterols, dietary phytostanols reduced phytosterol levels in plasma, red blood cells, and kidney, as well as altered mRNA levels of hepatic Abca1,Cyp27a1, and Hmgcr and intestinal Abcg5/8, Hmgcr and Srebp2. CONCLUSIONS: Altered expression of multiple sterol-regulatory genes may contribute to the incorporation and cholesterol-lowering actions of phytosterols and phytostanols. Phytosterols and phytostanols may act through different mechanism(s) on cholesterol and phytosterol/phytostanol trafficking.

Inhibition of cholesterol transport into skin cells in cultures by phytosterol-loaded microemulsion.

Cholesterol and plant phytosterols are lipophilic compounds solubilized by intestinal micelles in a competitive manner. In this work, we used radioactive cholesterol- and phytosterol-loaded oil-in-water microemulsions to follow their incorporation and mutual competition in HaCaT keratinocytes, SZ95 sebocytes, and skin pieces in cultures. Dynamic light scattering showed homogenous nanostructures of 10.5+/-1.5 nm diameter and cryo-transmission electron microscopy confirmed the presence of uniform spherical droplets of 7.0+/-1.0 nm diameter. Up to 320 nmol/ml of cholesterol can be solubilized and transported into cells with minimal toxic effect by 0.5 wt% nanodroplets in a cell medium. Phytosterols inhibit incorporation of cholesterol into cells, in vitro, at molar ratios (phytosterols/cholesterol) of 4 and above. The loaded nanodroplets accumulate in intracellular vesicles (presumably endosomes). No metabolic conversion of cholesterol or phytosterols was found in these cells, in vitro, after 24 h, at 37 degrees C.

Sinugrandisterols A-D, trihydroxysteroids from the soft coral Sinularia grandilobata.

Four new trihydroxysteroids, sinugrandisterols A-D (1-4), have been isolated from the CH(2)Cl(2)-soluble fraction of the EtOH extract of Sinularia grandilobata. The structures of these metabolites were determined on the basis of spectroscopic (IR, MS, and 1D and 2D NMR) analysis. The cytotoxicity of 1-4 toward a limited panel of cancer cell lines is also reported.

Ganoderma total sterol (GS) and GS1 protect rat cerebral cortical neurons from hypoxia/reoxygenation injury.

The effect of Ganoderma total sterol (GS) and its main components(GS(1)) on rat cortical neuronal cultures exposed to hypoxia/reoxygenation (H/R) was studied in vitro. GS (0.01,0.1,1 microg/ml) increased neuron viability following H/R. GS also significantly reduced malondialdehyde content and reactive oxygen species production and increased manganese superoxide dismutase (Mn-SOD) activity; furthermore, the translocation of nuclear factor-kappa B and the production of interleukin-1beta and tumor necrosis factor alpha induced by H/R were also blocked. These findings suggest that GS might be useful in treating H/R-induced oxidative stress and inflammatory response. We also hypothesized that Mn-SOD might play a critical role in the neuroprotective effect of GS against H/R injury. In addition, pretreatment with GS(1) (0.01, 0.1, 1 microg/ml) significantly attenuated the decline of neuron viability and the formation of reactive oxygen species. Furthermore GS(1) possessed more potent protective effect on neurons compared with GS at the same dose. These findings demonstrated that GS(1) is the main component in GS; and play a critical role in the neuroprotective effect of GS against H/R.

Long-term effects of sterol depletion in C. elegans: sterol content of synchronized wild-type and mutant populations.

Three major long-term effects of sterol deprivation in Caenorhabditis elegans are described. 1) The life expectancy of sterol-deprived wild-type animals is decreased by more than 40%. Similar decreases are found in animals carrying mutations in the daf-9, daf-12, daf-16, and clk-1 genes, suggesting that previously described aging pathways involving these genes are not involved in the life-extending effects of sterols. 2) There is a premature loss of motility, measured by response to mild touch. 3) There is a rapid postreproductive onset of sarcopenia (muscle wasting) as measured by total body fluorescence in a myo3::GFP-expressing strain. We also report that five sterols (the desmethylsterols cholesterol, 7-dehydrocholesterol, and lathosterol and the 4alpha-methyl sterols lophenol and 4alpha-methyl-cholesta-Delta8(14)-en-3beta-ol) are found in significant amounts at all stages of development and aging in cholesterol-fed animals. Supplying any one of these as the sole sterol confers similar protection from the long-term effects of sterol deprivation. These findings suggest that sterols are required continuously throughout the animal's life.

Interactions between sterol biosynthesis genes in embryonic development of Arabidopsis.

The sterol biosynthesis pathway of Arabidopsis produces a large set of structurally related phytosterols including sitosterol and campesterol, the latter being the precursor of the brassinosteroids (BRs). While BRs are implicated as phytohormones in post-embryonic growth, the functions of other types of steroid molecules are not clear. Characterization of the fackel (fk) mutants provided the first hint that sterols play a role in plant embryogenesis. FK encodes a sterol C-14 reductase that acts upstream of all known enzymatic steps corresponding to BR biosynthesis mutants. Here we report that genetic screens for fk-like seedling and embryonic phenotypes have identified two additional genes coding for sterol biosynthesis enzymes: CEPHALOPOD (CPH), a C-24 sterol methyl transferase, and HYDRA1 (HYD1), a sterol C-8,7 isomerase. We describe genetic interactions between cph, hyd1 and fk, and studies with 15-azasterol, an inhibitor of sterol C-14 reductase. Our experiments reveal that FK and HYD1 act sequentially, whereas CPH acts independently of these genes to produce essential sterols. Similar experiments indicate that the BR biosynthesis gene DWF1 acts independently of FK, whereas BR receptor gene BRI1 acts downstream of FK to promote post-embryonic growth. We found embryonic patterning defects in cph mutants and describe a GC-MS analysis of cph tissues which suggests that steroid molecules in addition to BRs play critical roles during plant embryogenesis. Taken together, our results imply that the sterol biosynthesis pathway is not a simple linear pathway but a complex network of enzymes that produce essential steroid molecules for plant growth and development.

Progestins block cholesterol synthesis to produce meiosis-activating sterols.

The resumption of meiosis is regulated by meiosis-preventing and meiosis-activating substances in testes and ovaries. Certain C29 precursors of cholesterol are present at elevated levels in gonadal tissue, but the mechanism by which these meiosis-activating sterols (MAS) accumulate has remained an unresolved question. Here we report that progestins alter cholesterol synthesis in HepG2 cells and rat testes to increase levels of major MAS (FF-MAS and T-MAS). These C29 sterols accumulated as a result of inhibition of Delta24-reduction and 4alpha-demethylation. Progesterone, pregnenolone, and 17alpha-OH-pregnenolone were potent inhibitors of Delta24-reduction in an in vitro cell assay and led to the accumulation of desmosterol, a Delta5,24 sterol precursor of cholesterol. A markedly different effect was observed for 17alpha-OH-progesterone, which caused the accumulation of sterols associated with inhibition of 4alpha-demethylation. The flux of 13C-acetate into lathosterol and cholesterol was decreased by progestins as measured by isotopomer spectral analysis, whereas newly synthesized MAS accumulated. The combined evidence that MAS concentrations can be regulated by physiological levels of progestins and their specific combination provides a plausible explanation for the elevated concentration of MAS in gonads and suggests a new role for progestins in fertility.