Effects of Long-Term High-Ergosterol Intake on the Cholesterol and Vitamin D Biosynthetic Pathways of Rats Fed a High-Fat and High-Sucrose Diet.

Dyslipidemia is a lifestyle-related (physical inactivity or obesity) disease; therefore, dietary foods that can easily be consumed in daily life is important to prevent dyslipidemia. Ergosterol, a precursor of vitamin D2, is a fungal sterol present in the membranes of edible mushrooms and other fungi. Ergosterol is converted to brassicasterol by 7-dehydrocholesterol reductase (DHCR7), a cholesterol biosynthesis enzyme that converts 7-dehydrocholesterol (a precursor of vitamin D3) into cholesterol. Previously, we reported that ergosterol increases 7-dehydrocholesterol, decreases cholesterol levels by competitive effect of DHCR7, and reduces DHCR7 mRNA and protein levels in human HepG2 hepatoma cells. Here, we investigated the effects of long-term high ergosterol intake on the cholesterol, vitamin D2, and D3 biosynthetic pathways of rats fed a high-fat and high-sucrose (HFHS) diet using GC-MS and LC with tandem mass spectrometry. In HFHS rats, oral ergosterol administration for 14 weeks significantly decreased plasma low-density lipoprotein cholesterol, total bile acid, and cholesterol precursor (squalene and desmosterol) levels and increased 7-dehydrocholesterol levels compared to HFHS rats without ergosterol. Ergosterol, brassicasterol, and vitamin D2 were detected, cholesterol levels were slightly decreased, and levels of vitamin D3 and its metabolites were slightly increased in rats fed HFHS with ergosterol. These results showed that ergosterol increased vitamin D2 levels, inhibited the cholesterol biosynthetic pathway, and possibly promoted vitamin D3 biosynthesis in vivo. Therefore, daily ergosterol intake may aid in the prevention of dyslipidemia.

Hypoxia-adapted Multiple Myeloma Stem Cells Resist γδ-T-Cell-mediated Killing by Modulating the Mevalonate Pathway.

BACKGROUND/AIM: The prognosis of patients with multiple myeloma (MM) has recently improved due to the emergence of new molecular targeting agents. However, MM remains incurable because MM stem cells are resistant to these agents. Therefore, it is essential to develop strategies to eradicate MM stem cells. We have previously demonstrated that MM cells cultured under prolonged hypoxic conditions (1% O2) (i.e., hypoxia-adapted MM cells; MM-HA cells) exhibited stem-cell-like characteristics. γδ T cells attack tumor cells by recognizing butyrophilin (BTN) 3A1 and BTN2A1, which are activated by the intracellular accumulation of isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway. In the present study, we investigated the cytotoxicity of γδ T cells against MM-HA stem-like cells. MATERIALS AND METHODS: We used a combination of flow cytometry, liquid chromatography-tandem mass spectrometry, and western blotting methods to investigate the cytotoxicity of γδ T cells against MM-HA cells and measured the amounts of IPP in MM-HA cells and their supernatants. RESULTS: The cytotoxicity of γδ T cells against MM-HA cells was significantly lower than that against MM cells cultured under normoxic conditions (20% O2; MM-Normo). Furthermore, the concentration of IPP in MM-HA cells was lower than that in MM-Normo cells. The expression of mevalonate decarboxylase and farnesyl diphosphate synthase proteins were decreased in MM-HA-cells. CONCLUSION: The cytotoxicity of γδ T cells against MM-HA cells was suppressed by the reduced IPP accumulation by modulating the mevalonate pathway in MM-HA cells.

Ergosterol increases 7-dehydrocholesterol, a cholesterol precursor, and decreases cholesterol in human HepG2 cells.

Current treatment approaches for hyperlipidemia rely mainly on reducing the cholesterol level by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which is involved in the presqualene pathway of cholesterol biosynthesis. Finding a compound that instead targets the postsqualene pathway could aid in the treatment of hyperlipidemia and synergistically reduce the cholesterol level when used in conjunction with HMGCR inhibitors. Ergosterol is a fungal sterol that is converted to brassicasterol by 7-dehydrocholesterol reductase (DHCR7). DHCR7 is also a cholesterol biosynthesis enzyme, and thus ergosterol may cause the accumulation of 7-dehydrocholesterol, a precursor of cholesterol and vitamin D3 , by a competitive effect. In this study, we examined the effect of ergosterol on the postsqualene pathway by quantifying cholesterol precursors and related sterols using gas chromatography-mass spectrometry and by conducting quantitative RT-PCR and western blot analysis for human HepG2 hepatoma cells. We found that ergosterol is converted into brassicasterol by the action of DHCR7 from HepG2 cells and that it induced the accumulation of cholesterol precursors (lathosterol, 7-dehydrocholesterol, and desmosterol) and decreased the cholesterol level by altering the mRNA and protein levels of cholesterol biosynthesis enzymes (increase of sterol 8,7-isomerase [EBP] and decrease of DHCR7 and 24-dehydrocholesterol reductase [DHCR24]). These results demonstrate that ergosterol inhibits the postsqualene pathway and may be useful for the prevention of hyperlipidemia.

New Inhibitory Effect of Latilactobacillus sakei UONUMA on the Cholesterol Biosynthesis Pathway in Human HepG2 Cells.

Many pharmaceuticals and dietary foods have been reported to inhibit cholesterol biosynthesis, mainly by inhibiting the presqualene enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase rather than a postsqualene enzyme. In this study, we examined the inhibitory effects of Latilactobacillus sakei UONUMA on cholesterol biosynthesis, especially postsqualene, in human HepG2 hepatoma cells. We quantified cholesterol and its precursors, and the mRNA and protein levels of enzymes involved in cholesterol biosynthesis. Three L. sakei UONUMA strains exhibited new inhibitory effects on cholesterol biosynthesis and inhibited the mRNA level of sterol-delta24-reductase (DHCR24), which is involved in the postsqualene cholesterol biosynthesis pathway. These strains will be useful for the prevention and treatment of hyperlipidemia.

Evaluating the effect of aromatherapy on a stress marker in healthy subjects.

BACKGROUND/PURPOSE: Chemotherapy is important for cancer treatment, but patients' physical and mental stress may lead to unfavorable pain control, an increase in the risk of relapse, and a reduction in the quality of life (QOL). Recently, aromatherapy has been performed in addition to palliative care in many countries, such as Japan and the United States, but scientific evidence remains insufficient. To investigate the usefulness of aromatherapy as complementary and alternative medicine, we evaluated its influence on the immune and autonomic nervous systems. METHODS: We instructed healthy volunteers to inhale aroma oil at bedtime for 6 weeks, and measured changes in the salivary level of secretory immunoglobulin A (s-IgA). Furthermore, blood was collected in addition to saliva in some healthy volunteers, and the blood level of noradrenaline (NA) was measured to examine its relationship to changes in the salivary s-IgA level. RESULTS: Aromatherapy with lavender and grapefruit oils significantly increased the salivary s-IgA level: lavender oil increased 3.5-fold (p = 0.03), and grapefruit oil increased 2.55-fold (p = 0.04). On lavender oil inhalation, there was a weak, positive correlation between changes in the salivary s-IgA level and those in the blood NA level (R2 = 0.24). CONCLUSION: The results showed that aromatherapy with lavender and grapefruit oils reduced stress by acting on the immune and autonomic nervous systems in healthy volunteers. In the future, its clinical usefulness must be investigated through similar examination in patients in whom the stress level may be high.

Isopentenyl pyrophosphate secreted from Zoledronate-stimulated myeloma cells, activates the chemotaxis of γδT cells.

γδT cell receptor (TCR)-positive T cells, which control the innate immune system, display anti-tumor immunity as well as other non-immune-mediated anti-cancer effects. γδT cells expanded ex vivo by nitrogen-containing bisphosphonate (N-BP) treatment can kill tumor cells. N-BP inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, resulting in the accumulation of isopentenyl pyrophosphate (IPP), which is a stimulatory antigen for γδT cells. We have previously observed that as they get closer, migrating γδT cells increase in speed toward target multiple myeloma (MM) cells. In the present study, we investigated the γδT cell chemotactic factors involving using a micro total analysis system-based microfluidic cellular analysis device. The addition of supernatant from RPMI8226 MM cells treated with the N-BP zoledronic acid (ZOL) or the addition of IPP to the device induced chemotaxis of γδT cells and increased the speed of migration compared to controls. Analysis of the ZOL-treated RPMI8226 cell supernatant revealed that it contained IPP secreted in a ZOL-dose-dependent manner. These observations indicate that IPP activates the chemotaxis of γδT cells toward target MM cells treated with ZOL.

Effect of polyphenols on 3-hydroxy-3-methylglutaryl-coenzyme A lyase activity in human hepatoma HepG2 cell extracts.

When carbohydrate metabolism is impaired, fatty acid metabolism is activated. Excess acetyl-coenzyme A (CoA) is generated from fatty acids by ß-oxidation and is used for the formation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) and subsequently for acetoacetate. High levels of secreted ketone bodies (acetoacetate and 3ß-hydroxybutyrate) lower the pH of blood and urine, resulting in ketoacidosis. HMG-CoA lyase in hepatic cells is a rate-limiting enzyme catalyzing the cleavage of HMG-CoA to acetoacetate, and thus inhibition of this enzyme results in reduced acetoacetate production, in other words, impaired ketoacidosis. Inhibition of HMG-CoA lyase activity possibly prevents ketoacidosis and should be the therapeutic target. Polyphenols are common and abundant dietary constituents with beneficial effects on human health. We examined the inhibitory effects of dietary polyphenols on HMG-CoA lyase activity in cellular extracts of human hepatoma HepG2 cells. Of the nine representative dietary polyphenols tested, (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), and gallic acid (GA) effectively inhibited HMG-CoA lyase activity. Lineweaver-Burk analysis revealed that EGC and EGCG are likely to be mixed-type noncompetitive inhibitors. Pyrogallol with the gallyl structure also inhibited HMG-CoA lyase activity, suggesting that the gallyl moiety of polyphenols is important for the inhibition of HMG-CoA lyase activity.

Inhibitory effect of protopanaxatriol ginseng metabolite M4 on the production of corticosteroids in ACTH-stimulated bovine adrenal fasciculata cells.

AIMS: We investigated the pharmacological effects of saponins isolated from ginseng root and their metabolites, which occur by hydrolysis of the sugar moieties connecting the aglycone of saponins in the digestive tract, on the production of corticosteroids in bovine adrenal fasciculata cells in vitro. MAIN METHODS: The levels of corticosteroids produced from adrenal corticotropic hormone (ACTH)-stimulated bovine adrenal fasciculata cells were determined under the presence or absence of ginseng saponins (ginsenosides) and their metabolites using fluorometry, gas-chromatography-mass spectrometry, and sweeping-micellar electrokinetic capillary chromatography. KEY FINDINGS: An end metabolite of the protopanaxatriol saponins in ginseng, 20(s)-protopanaxatriol (M4), strongly reduced ACTH-stimulated cortisol production. M4 significantly inhibited the production of cortisol induced by different stimuli, alamethicin, dibutyryl cyclic AMP, forskolin, and 22(R)-hydroxycholesterol, a membrane-permeable cholesterol. However, it did not affect the production of cortisol by either pregnenolone, a precursor of cortisol synthesis, or cyclic AMP. Furthermore, M4 significantly inhibited the production of pregnenolone, progesterone, deoxycorticosterone, cortisol, and corticosterone in a dose-dependent manner. SIGNIFICANCE: Results strongly suggest that protopanaxatriol saponins in ginseng are prodrugs metabolized in the digestive tract so that the end metabolite, M4, produces inhibitory activity of corticosteroid production in the adrenal fasciculata cells in vivo. The results also suggest that M4 inhibits the conversion from cholesterol to pregnenolone because the production of pregnenolone was reduced.

Effect of polyphenols on production of steroid hormones from human adrenocortical NCI-H295R cells.

Modulating steroid hormone levels is a curative and preventive measure for Cushing's syndrome, aldosteronism, and various stress-triggered symptoms. Polyphenols have been reported to inhibit steroidogenic enzymes such as 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and aromatase. However, evidence for their inhibitory effects is fragmentary because it has been determined in studies with small groups of steroid hormones. To investigate the effects of steroids on complete steroidogenic pathways, comprehensive analysis of steroid hormones is necessary. Here we cultured forskolin-stimulated NCI-H295R, a human adrenocortical carcinoma cell line, in the presence of a polyphenol and employed GC-MS to simultaneously determine the levels of nine steroid hormones (pregnenolone, progesterone, deoxycorticosterone, aldosterone, 17α-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, and estradiol) in cell culture supernatant. We found that daidzein, genistein, apigenin, hesperetin, naringenin, and eriodictyol significantly reduced deoxycorticosterone and androstenedione levels (p<0.05), suggesting inhibition of 3ß-HSD by these polyphenols. Apigenin was more potent than other polyphenols in increasing the levels of pregnenolone and 17α-hydroxyprogesterone, suggesting that it inhibits cytochrome P450 (CYP) 17 and CYP21, as well as 3ß-HSD. Real-time reverse transcription polymerase chain reaction showed that apigenin significantly downregulated the expression levels of 3ß-HSD, CYP17, and CYP21 mRNA (p<0.05). This is the first study to demonstrate the inhibitory effects of apigenin on CYP17 and CYP21.

Oxysterol changes along with cholesterol and vitamin D changes in adult phenylketonuric patients diagnosed by newborn mass-screening.

BACKGROUND: Phenylketonuria (PKU) possibly leads to hypocholesterolemia and lowered vitamin D (VD) status. Metabolism of oxysterols linking with those of cholesterol and VD has never been examined in PKU. METHODS: Blood oxysterols along with blood phenylalanine, lipids and VD were examined for 33 PKU adults aged 21-38 years and 20 age-matched healthy controls. RESULTS: Total- and low-density cholesterols, and 25-hydroxy VD(3) were decreased significantly in the PKU group (cholesterols, 10% decrease; 25-hydroxy VD(3) 35% decrease vs. the control group). 24S-hydroxycholesterol (24S-OHC) eliminating brain cholesterol, and 27-OHC and 7α-hydroxycholesterol (7α-OHC) representing peripheral and hepatic cholesterol elimination, respectively, were significantly decreased in PKU group: 24S-OHC, 25% decrease, p<.01; 27-OHC and 7α-OHC, 35-40% decrease, p<.001. 7ß-Hydroxycholesterol (7ß-OHC) reflecting oxidative stress was increased significantly in PKU group (p<.05). 7α-OHC and 27-OHC levels in PKU group always showed similar values, regardless of other parameters while the 24S-OHC and 7ß-OHC levels decreased and increased, respectively, showing significant correlations with phenylalanine level (p<.005). 27-OHC level showed a significant positive correlation with the 25-hydroxy VD(3) level in this group (p<.001). CONCLUSION: Blood oxysterol changes predominate over blood cholesterol changes and influence on VD status in adult PKU patients.

Simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by semi-micro high-performance liquid chromatography with an immobilized cholesterol oxidase as a pre-column reactor: application to bovine adrenal fasciculata cells.

A method for the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by high-performance liquid chromatography with an immobilized cholesterol oxidation enzyme reactor was developed. Pregnenolone and 17α-hydroxypregnenolone were converted to progesterone and 17α-hydroxyprogesterone, respectively, by the immobilized enzyme packed into the reactor column, and could thus be monitored by UV absorption at 240 nm. The calibration curves for pregnenolone and 17α-hydroxypregnenolone were linear in the range of 0.4-10 and 0.3-10 µg/ml with a correlation coefficient of 0.9993 and 0.9998, respectively. The detection limit at a signal-to-noise ratio of 3 was 0.12 and 0.08 µg/ml for pregnenolone and 17α-hydroxypregnenolone, respectively. The conversion rate of pregnenolone to progesterone and 17α-hydroxypregnenolone to 17α-hydroxyprogesterone was 90.6% and 99.3%, respectively. Intra-day and inter-day precision (in terms of percentage coefficient of variation) were less than 9.3%, with accuracy greater than 94.8%. This method was successfully applied to the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone secreted into the culture medium of bovine adrenal fasciculata cells and of both analytes produced within the cells.

Inhibitory effect of antimicrobial agents and anisodamine on the staphylococcal superantigenic toxin-induced overproduction of proinflammatory cytokines by human peripheral blood mononuclear cells.

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), produces superantigenictoxins, such as toxic shock syndrome toxin-1 (TSST-1). TSST-1 abnormally activates T cells to overproduce inflammatory cytokines (such as tumor necrosis factor-alpha, interleukin-2, and interferon-gamma) leading to shock. In this study, we investigated the inhibitory effect of antimicrobial agents and anisodamine (a Chinese herbal extract) on TSST-1-induced cytokine production. Among the macrolides and related agents examined, azithromycin and rokitamycin showed the greatest inhibitory activity against the TSST-1-induced cytokine production. This inhibitory effect was similar to that of anisodamine, which, however, had no inhibitory activity against bacterial growth. Vancomycin, teicoplanin, arbekacin, and linezolid (anti-MRSA and related agents) had no significant inhibitory effect on cytokine production. The inhibitory effect of the drugs on cell proliferation was not significant. These data indicate that some antimicrobial agents, e.g., azithromycin and rokitamycin, manifest anti-superantigenic toxin activity through the inhibition of cytokine production, just like anisodamine.

Effects of azithromycin on shiga toxin production by Escherichia coli and subsequent host inflammatory response.

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the human intestinal mucosa, produces Stx from phage, and causes the development of hemolytic-uremic syndrome via Stx-induced inflammatory cytokine production. Azithromycin exhibited strong in vitro activity against STEC without inducing Stx-converting phage, in marked contrast to norfloxacin. Azithromycin decreased the tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 production from Stx-treated human peripheral mononuclear cells or monocytes to a greater extent than did clarithromycin. In Stx-injected mice, azithromycin significantly suppressed Stx-induced TNF-alpha, IL-1beta, and IL-6 levels in serum and improved the outcome as assessed by survival rate. In the STEC oral infection experiment using immature mice immediately after weaning (weaned immature-mouse model), all mice died within 7 days postinfection. Azithromycin administration gave the mice 100% protection from killing, while ciprofloxacin administration gave them 67% protection. The data suggest that azithromycin (at least at higher concentrations) has a strong effect on Stx production by STEC and on the Stx-induced inflammatory host response and prevents death in mice. Azithromycin may have a beneficial effect on STEC-associated disease.