Pharmacological inhibition of acyl-coenzyme A:cholesterol acyltransferase alleviates obesity and insulin resistance in diet-induced obese mice by regulating food intake.

BACKGROUND/OBJECTIVES: Acyl-coenzyme A:cholesterol acyltransferases (ACATs) catalyze the formation of cholesteryl ester (CE) from free cholesterol to regulate intracellular cholesterol homeostasis. Despite the well-documented role of ACATs in hypercholesterolemia and their emerging role in cancer and Alzheimer's disease, the role of ACATs in adipose lipid metabolism and obesity is poorly understood. Herein, we investigated the therapeutic potential of pharmacological inhibition of ACATs in obesity. METHODS: We administrated avasimibe, an ACAT inhibitor, or vehicle to high-fat diet-induced obese (DIO) mice via intraperitoneal injection and evaluated adiposity, food intake, energy expenditure, and glucose homeostasis. Moreover, we examined the effect of avasimibe on the expressions of the genes in adipogenesis, lipogenesis, inflammation and adipose pathology in adipose tissue by real-time PCR. We also performed a pair feeding study to determine the mechanism for body weight lowering effect of avasimibe. RESULTS: Avasimibe treatment markedly decreased body weight, body fat content and food intake with increased energy expenditure in DIO mice. Avasimibe treatment significantly lowered blood levels of glucose and insulin, and improved glucose tolerance in obese mice. The beneficial effects of avasimibe were associated with lower levels of adipocyte-specific genes in adipose tissue and the suppression of food intake. Using a pair-feeding study, we further demonstrated that avasimibe-promoted weight loss is attributed mainly to the reduction of food intake. CONCLUSIONS: These results indicate that avasimibe ameliorates obesity and its-related insulin resistance in DIO mice through, at least in part, suppression of food intake.

Perilipin 5 alleviates HCV NS5A-induced lipotoxic injuries in liver.

BACKGROUND: The homeostasis of lipid droplets (LDs) plays a crucial role in maintaining the physical metabolic processes in cells, and is regulated by many LD-associated proteins, including perilipin 5 (Plin5) in liver. As the putative sites of hepatitis C virus (HCV) virion assembly, LDs are vital to viral infection. In addition, the hepatic LD metabolism can be disturbed by non-structural HCV proteins, such as NS5A, but the details are still inexplicit. METHODS: HCV NS5A was overexpressed in the livers and hepatocytes of wild-type and Plin5-null mice. BODIPY 493/503 and oil red O staining were used to detect the lipid content in mouse livers and hepatocytes. The levels of lipids, lipid peroxidation and inflammation biomarkers were further determined. Immunofluorescence assay and co-immunoprecipitation assay were performed to investigate the relationship of Plin5 and NS5A. RESULTS: One week after adenovirus injection, livers expressing NS5A showed more inflammatory cell aggregation and more severe hepatic injuries in Plin5-null mice than in control mice, which was consistent with the increased serum levels of IL-2 and TNF-α (P < 0.05) observed in Plin5-null mice. Moreover, Plin5 deficiency in the liver and hepatocytes aggravated the elevation of MDA and 4-HNE levels induced by NS5A expression (P < 0.01). The triglyceride (TG) content was increased approximately 25% by NS5A expression in the wild-type liver and hepatocytes but was unchanged in the Plin5-null liver and hepatocytes. More importantly, Plin5 deficiency in the liver and hepatocytes exacerbated the elevation of non-esterified fatty acids (NEFAs) stimulated by NS5A expression (P < 0.05 and 0.01 respectively). Using triacsin C to block acyl-CoA biosynthesis, we found that Plin5 deficiency aggravated the NS5A-induced lipolysis of TG. In contrast, Plin5 overexpression in HepG2 cells ameliorated the NS5A-induced lipolysis and lipotoxic injuries. Immunofluorescent staining demonstrated that NS5A expression stimulated the targeting of Plin5 to the surface of the LDs in hepatocytes without altering the protein levels of Plin5. By co-IP, we found that the N-terminal domain (aa 32-128) of Plin5 was pivotal for its binding with NS5A. CONCLUSIONS: Our data highlight a protective role of Plin5 against hepatic lipotoxic injuries induced by HCV NS5A, which is helpful for understanding the steatosis and injuries in liver during HCV infection.

Timing of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor initiation and allograft vasculopathy progression and outcomes in heart transplant recipients.

AIMS: Early studies from the 1990s have shown that statins improve survival and attenuate cardiac allograft vasculopathy (CAV). However, little contemporary data are available on the incremental benefit of statins with the current use of new-generation immunosuppressive agents and the use of coronary intravascular ultrasound for assessment of CAV. We sought to investigate the effect of early statin (ES) as compared with late statin (LS) initiation after heart transplantation (HT) on long-term CAV progression and clinical outcomes in a large contemporary HT cohort. METHODS AND RESULTS: We analysed a cohort of 409 adult HT recipients. CAV progression was assessed by serial coronary intravascular ultrasound volumetric measurements of the differences between baseline and last follow-up plaque volume (PV) and plaque index (PV/vessel volume ratio). CAV progression and clinical outcomes were compared between the ES (<2 years after HT) and the LS (>2 years after HT) groups. During a median follow-up of 8.2 years, ES resulted in significantly lower change (Δ) of plaque index (+3.8% ± 1.7% vs. +8.2% ± 3.6%; P = 0.0008) and PV (+0.8 ± 0.3 vs. +1.9 ± 1.2; P = 0.045) compared with LS group. In a Cox proportional hazards regression model and after adjustment for baseline characteristics, ES was associated with a 52% decreased risk of CAV-associated events (hazard ratio 0.48, 95% confidence interval: 0.27-0.91; P = 0.025) and a 42% decreased risk of the composite endpoint of all-cause mortality and CAV-associated events (hazard ratio 0.58, 95% confidence interval: 0.38-0.91; P = 0.019). CONCLUSIONS: Early initiation of statin therapy after HT results in attenuated CAV progression as well as in decreased CAV-related events and mortality.

Lovastatin analogues and other metabolites from soil-derived Aspergillus terreus YIM PH30711.

Eight previously undescribed metabolites including of lovastatin analogues, a pair of diastereoisomers, a cyclopentenone dimer, and three polyketides were isolated from the culture of Aspergillus terreus YIM PH30711. Two types of unprecedented skeletons, benzene-cyclopentanone complex and linear polyketide, and an unusual dimer structure were determined by spectral analysis. Compound, 3α-hydroxy-3,5-dihydromonacolin L showed moderate activity against HMG-CoA reductase, with an inhibition ratio of 34% at the concentration of 50 µM, while lovastatin and dihydromonacolin K ethyl ester presented much stronger activity against HMGR with inhibition rates of 85% and 90% at the concentration of 50 µM, respectively. Aspereusin A was active against AChE with a ratio of 62% at the concentration of 50 µM, while its stereomers did not showed obvious inhibition (<10%). The configuration at C-4 of these three diastereoisomers was crucial in the inhibition against AChE, and the ß-orientation of substituted methoxyl acrylic acid should be beneficial to the combining with AChE.

Simvastatin plus capecitabine-cisplatin versus placebo plus capecitabine-cisplatin in patients with previously untreated advanced gastric cancer: a double-blind randomised phase 3 study.

PURPOSE: We aimed to the addition of synthetic 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase inhibitor, simvastatin to capecitabine-cisplatin (XP) in patients with previously untreated advanced gastric cancer (AGC). METHODS: In this double-blind, placebo-controlled, phase III study, we enrolled patients aged 18 years or older with histological or cytological confirmed metastatic adenocarcinoma of the stomach or gastroesophageal junction (GEJ) at nine centres in Korea. Patients, stratified by disease measurability and participating site, were randomly assigned (1:1) to receive capecitabine 1000mg/m(2) twice daily for 14 days and cisplatin 80 mg/m(2) on day 1 every 3 weeks plus either simvastatin 40 mg or placebo, once daily. Cisplatin was given for 8 cycles; capecitabine and simvastatin were administered until disease progression or unacceptable toxicities. This study is registered with ClinicalTrials.gov, number NCT01099085. RESULTS: Between February 2009 and November 2012, 244 patients were enrolled and assigned to treatment groups (120 simvastatin, 124 placebo). Median progression free survival (PFS) for 120 patients allocated XP plus simvastatin was 5.2 months (95% confidence interval (CI) 4.3-6.1) compared with 4.63 months (95% CI 3.5-5.7) for 124 patients who were allocated to XP plus placebo (hazard ratio 0.930, 95% CI 0.684-1.264; p=0.642). 63 (52.5%) of 120 patients in simvastatin group and 70 (56.4%) of 124 had grade 3 or higher adverse events. CONCLUSIONS: Addition of 40 mg simvastatin to XP does not increase PFS in our trial, although it does not increase toxicity. Low dose of simvastatin (40 mg) to chemotherapy is not recommended in untargeted population with AGC.

Immune-mediated myopathy related to anti 3-hydroxy-3-methylglutaryl-coenzyme A reductase antibodies as an emerging cause of necrotizing myopathy induced by statins.

Immune-mediated necrotizing myopathy (IMNM) associated with statin use and anti 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibody is a new and emerging entity that supports a link between statin use and IMNM and raises the questions of distinct clinical phenotypes and treatment strategy. We describe the clinical and histopathological characteristics of a patient and discuss the spectrum of IMNM and statin-induced myopathies. A 65-year-old man was suffering from proximal muscle weakness and elevated CK levels, following exposure to statin therapy. The symptoms worsened despite discontinuation of the drug. At that point, no myositis-specific or -associated antibodies were detected. Malignancy screening did not reveal abnormalities. Muscle biopsy demonstrated a predominantly necrotizing myopathy with minimal lymphocytic infiltrates, MHC class I expression in necrotic muscle fibers, and complement deposition on scattered non-necrotic muscle fibers. Muscle protein analysis by western blot was normal. The patient did not improve with steroid and methotrexate and required monthly intravenous immunoglobulin (IVIG) therapy. Muscle strength gradually improved, CK levels normalized and IVIG were stopped 1 year later. Screening for anti-HMGCR antibodies, not available at the time of presentation, was highly positive. Identification of anti-HMGCR antibodies in statin-exposed patients with myopathy appears to be helpful both for differential diagnosis and for treatment strategy. In patients who did not improve after discontinuation of the statin treatment, a muscle biopsy should be performed as well as screening for anti-HMGCR antibodies. Patients with this disorder require aggressive immunosuppressive treatment.

Luminol electrochemiluminescence for the analysis of active cholesterol at the plasma membrane in single mammalian cells.

A luminol electrochemiluminescence assay was reported to analyze active cholesterol at the plasma membrane in single mammalian cells. The cellular membrane cholesterol was activated by the exposure of the cells to low ionic strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT). The active membrane cholesterol was reacted with cholesterol oxidase in the solution to generate a peak concentration of hydrogen peroxide on the electrode surface, which induced a measurable luminol electrochemiluminescence. Further treatment of the active cells with mevastatin decreased the active membrane cholesterol resulting in a drop in luminance. No change in the intracellular calcium was observed in the presence of luminol and voltage, which indicated that our analysis process might not interrupt the intracellular cholesterol trafficking. Single cell analysis was performed by placing a pinhole below the electrode so that only one cell was exposed to the photomultiplier tube (PMT). Twelve single cells were analyzed individually, and a large deviation on luminance ratio observed exhibited the cell heterogeneity on the active membrane cholesterol. The smaller deviation on ACAT/HMGCoA inhibited cells than ACAT inhibited cells suggested different inhibition efficiency for sandoz 58035 and mevastatin. The new information obtained from single cell analysis might provide a new insight on the study of intracellular cholesterol trafficking.

HMG-CoA reductase inhibitors induce apoptosis of lymphoma cells by promoting ROS generation and regulating Akt, Erk and p38 signals via suppression of mevalonate pathway.

Statins, the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are widely used cholesterol-lowering drugs. Convincing evidence indicates that statins stimulate apoptotic cell death in several types of proliferating tumor cells in a cholesterol-lowering-independent manner. The objective here was to elucidate the molecular mechanism by which statins induce lymphoma cells death. Statins (atorvastatin, fluvastatin and simvastatin) treatment enhanced the DNA fragmentation and the activation of proapoptotic members such as caspase-3, PARP and Bax, but suppressed the activation of anti-apoptotic molecule Bcl-2 in lymphoma cells including A20 and EL4 cells, which was accompanied by inhibition of cell survival. Both increase in levels of reactive oxygen species (ROS) and activation of p38 MAPK and decrease in mitochondrial membrane potential and activation of Akt and Erk pathways were observed in statin-treated lymphoma cells. Statin-induced cytotoxic effects, DNA fragmentation and changes of activation of caspase-3, Akt, Erk and p38 were blocked by antioxidant (N-acetylcysteine) and metabolic products of the HMG-CoA reductase reaction, such as mevalonate, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These results suggests that HMG-CoA reductase inhibitors induce lymphoma cells apoptosis by increasing intracellular ROS generation and p38 activation and suppressing activation of Akt and Erk pathways, through inhibition of metabolic products of the HMG-CoA reductase reaction including mevalonate, FPP and GGPP.

Simvastatin prevents proliferation and bone metastases of lung adenocarcinoma in vitro and in vivo.

OBJECTIVES: To explore the mechanism about how HMG-CoA reductase (HMGR) inhibitor inhibit proliferation and bone metastases of lung adenocarcinoma in vitro and in vivo. METHODS: The HMGR inhibitor simvastatin, human lung cancer cell line A549 and Balb/c nude mouse were used in this study. The mice were randomly divided into 2 groups: control group (0.9% NaCl solution, i.v.) and simvastatin group (5mg/kg simvastatin, i.v.). A scratch assay using A549 cell monolayer was also tested. An invasion assay using collagen-coated membrane in trans-wells was applied to evaluate the effect of simvastatin on the metastatic potential of A549 cells in vitro. The expressions of CD44, PUMA, P53, MMP2 and MMP9 were determined by real-time PCR and western blotting; the phosphorylation status of MAPK/ERK signaling parthway was investigated by western blot. . RESULTS: Compared with the control group, the migration of A549 cells in simvastatin-treated group was markedly inhibited (p ≤ 0.01). Untreated A549 cells showed marked invasion, while simvastatin significantly inhibited the invasion of tumor cells (p ≤ 0.001). Incubation of A549 cells with simvastatin significantly reduced the levels of CD44, MMP2 and MMP9 (p <0.01), while significantly increased p53 (p < 0.01). Simvastatin significantly inhibits tumor growth and bone metastasis in lung cancer xenograft mouse model, simvastatin can inhibit the kinase phosphorylation inMAPK/ERK signaling parthway. CONCLUSIONS: The HMGR inhibitor simvastatin prevents proliferation and osteolytic bone metastases of lung adenocarcinoma cells in vitro and vivo. Its mechanism may be associated with regulation of CD44, P53, MMP family and inactivation of MAPK/ERK signaling parthway.

Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and application of statins as a novel effective therapeutic approach against Acanthamoeba infections.

Acanthamoeba is an opportunistic pathogen in humans, whose infections most commonly manifest as Acanthamoeba keratitis or, more rarely, granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba, they are generally lengthy and/or have limited efficacy. Therefore, there is a requirement for the identification, validation, and development of novel therapeutic targets against these pathogens. Recently, RNA interference (RNAi) has been widely used for these validation purposes and has proven to be a powerful tool for Acanthamoeba therapeutics. Ergosterol is one of the major sterols in the membrane of Acanthamoeba. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, one of the precursors for the production of cholesterol in humans and ergosterol in plants, fungi, and protozoa. Statins are compounds which inhibit this enzyme and so are promising as chemotherapeutics. In order to validate whether this enzyme could be an interesting therapeutic target in Acanthamoeba, small interfering RNAs (siRNAs) against HMG-CoA were developed and used to evaluate the effects induced by the inhibition of Acanthamoeba HMG-CoA. It was found that HMG-CoA is a potential drug target in these pathogenic free-living amoebae, and various statins were evaluated in vitro against three clinical strains of Acanthamoeba by using a colorimetric assay, showing important activities against the tested strains. We conclude that the targeting of HMG-CoA and Acanthamoeba treatment using statins is a novel powerful treatment option against Acanthamoeba species in human disease.

[Microbial metabolites that inhibit sterol biosynthesis, their chemical diversity and characteristics of mode of action].

Inhibitors of sterol biosynthesis (ISB) are widespread in nature and characterized by appreciable diversity both in their chemical structure and mode of action. Many of these inhibitors express noticeable biological activity and approved themselves in development of various pharmaceuticals. In this review there is a detailed description of biologically active microbial metabolites with revealed chemical structure that have ability to inhibit sterol biosynthesis. Inhibitors of mevalonate pathway in fungous and mammalian cells, exhibiting hypolipidemic or antifungal activity, as well as inhibitors of alternative non-mevalonate (pyruvate gliceraldehyde phosphate) isoprenoid pathway, which are promising in the development of affective antimicrobial or antiparasitic drugs, are under consideration in this review. Chemical formulas of the main natural inhibitors and their semi-synthetic derivatives are represented. Mechanism of their action at cellular and biochemical level is discussed. Special attention is given to inhibitors of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase (group of lovastatin) and inhibitors of acyl-CoA-cholesterol-acyl transferase (ACAT) that possess hypolipidemic activity and could be affective in the treatment of atherosclerosis. In case of inhibitors of late stages of sterol biosynthesis (after squalene formation) special attention is paid to compounds possessing evident antifungal and antitumoral activity. Explanation of mechanism of anticancer and antiviral action of microbial ISB, as well as the description of their ability to induce apoptosis is given.

Effects of HMG-CoA reductase inhibitors on the pharmacokinetics of losartan and its main metabolite EXP-3174 in rats: possible role of CYP3A4 and P-gp inhibition by HMG-CoA reductase inhibitors.

The present study was designed to investigate the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (atorvastatin, pravastatin, simvastatin) on the pharmacokinetics of losartan and its active metabolite EXP-3174 in rats. Pharmacokinetic parameters of losartan and EXP-3174 in rats were determined after oral and intravenous administration of losartan (9 mg/kg) without and with HMG-CoA reductase inhibitors (1 mg/kg). The effect of HMG-CoA reductase inhibitors on P-gp and cytochrome (CYP) 3A4 activity were also evaluated. Atorvastatin, pravastatin and simvastatin inhibited CYP3A4 activities with IC50 values of 48.0, 14.1 and 3.10 µmol/l, respectively. Simvastatin (1-10 µmol/l) enhanced the cellular uptake of rhodamine-123 in a concentration-dependent manner. The area under the plasma concentration-time curve (AUC0₋∞) and the peak plasma concentration of losartan were significantly (p < 0.05) increased by 59.6 and 45.8%, respectively, by simvastatin compared to those of control. The total body clearance (CL/F) of losartan after oral administration with simvastatin was significantly decreased (by 34.8%) compared to that of controls. Consequently, the absolute bioavailability (F) of losartan after oral administration with simvastatin was significantly increased by 59.4% compared to that of control. The metabolite-parent AUC ratio was significantly decreased by 25.7%, suggesting that metabolism of losartan was inhibited by simvastatin. In conclusion, the enhanced bioavailability of losartan might be mainly due to inhibition of P-gp in the small intestine and CYP3A subfamily-mediated metabolism of losartan in the small intestine and/or liver and to reduction of the CL/F of losartan by simvastatin.

Regulation of stearoyl-coenzyme A desaturase and fatty acid delta-6 desaturase-2 expression by linoleic acid and arachidonic acid in human sebocytes leads to enhancement of proinflammatory activity but does not affect lipogenesis.

BACKGROUND: Treatment of SZ95 sebocytes with the essential fatty acid linoleic acid (LA) and the polyunsaturated fatty acid arachidonic acid (AA) leads to sebaceous lipogenesis. Animal data indicate that stearoyl-coenzyme A desaturase (SCD), a key enzyme in fatty acid biosynthesis, is involved in sebaceous lipogenesis and proinflammatory signalling in the sebaceous gland. On the other hand, fatty acid delta-6 desaturase-2 (FADS2) catalyses the conversion of LA to AA. OBJECTIVES: To identify the effects of LA and AA on the expression of SCD and FADS2 and to detect its biological relevance. METHODS: SZ95 sebocytes were treated with LA (10(-5) and 10(-4) mol L(-1) ), AA (10(-6) and 10(-5) mol L(-1) ) and the combination of LA (10(-4) mol L(-1) ) and testosterone (2 × 10(-8) mol L(-1) ), with or without addition of the SCD inhibitor FPCA (10(-8) and 10(-6) mol L(-1) ). Cytotoxicity was determined by the lactate dehydrogenase assay. SCD and FACS2 mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction and protein expression by Western blot analysis. SZ95 sebocyte lipid content and cell number were measured by the Nile red and the fluorescein diacetate microassays, respectively. Determination of interleukin (IL)-6 and IL-8 release was evaluated by enzyme-linked immunosorbent assay. RESULTS: LA treatment induced an increase of SCD and FADS2 at mRNA and protein levels in SZ95 sebocytes after 1·5 h. Treatment with AA led to an increase of SCD but to a decrease of FADS2 mRNA levels. LA/testosterone cotreatment stimulated lipogenesis in SZ95 sebocytes. A distinct proinflammatory pattern was registered: whereas LA strongly upregulated IL-6 secretion only, AA induced a mild level of IL-6 and IL-8 release from SZ95 sebocytes. Treatment with the SCD inhibitor FPCA reduced the LA/testosterone-upregulated SCD and FADS2 mRNA levels and resulted in an anti-inflammatory effect, but did not affect sebaceous lipogenesis. CONCLUSIONS: LA-induced sebaceous lipogenesis is likely to be an SCD-independent effect. Regulation of SCD and FADS2 expression by LA and AA leads to enhancement of proinflammatory activity but does not affect lipogenesis in human sebocytes.

Relevance of the mevalonate biosynthetic pathway in the regulation of bone marrow mesenchymal stromal cell-mediated effects on T-cell proliferation and B-cell survival.

BACKGROUND: Bone marrow mesenchymal stromal cells can suppress T-lymphocyte proliferation but promote survival of normal and malignant B cells, thus representing a possible target for new therapeutic schemes. Here we defined the effects of cholesterol synthesis inhibitors on the interaction between these mesenchymal stromal cells and T or B lymphocytes. DESIGN AND METHODS: We exposed mesenchymal stromal cells to inhibitors, such as fluvastatin, of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, responsible for the synthesis of mevalonate, the precursor of cholesterol. Also, these cells were treated with manumycin A, a farnesyl transferase inhibitor which blocks the mevalonate-dependent isoprenylation of small guanosin triphosphate binding proteins. First, mesenchymal stromal cell morphology, cytoskeleton assembly, cell cycle, survival and cytokine production were evaluated. Then, these cells were co-cultured with either T or B lymphocytes and we analyzed: 1) the inhibition of T-cell proliferation to mitogenic stimuli; 2) B-cell survival. RESULTS: Fluvastatin altered the assembly of actin microfilaments, inactivated RhoA guanosin triphosphate binding protein, inhibited the S-phase of the cell cycle, induced apoptosis in a small fraction of cells but preserved cytokine production. Preincubation of mesenchymal stromal cells with fluvastatin, or manumycin A, down-regulated the expression of adhesion molecules, reduced cell-to-cell interactions and prevented the inhibition exerted by these stromal cells on CD3/T-cell receptor-induced lymphocyte proliferation. Mevalonic acid could revert morphological, phenotypic and functional effects of fluvastatin. Finally, fluvastatin significantly reduced the mesenchymal stromal cells-mediated rescue of B cells in the presence of dexamethasone, although it did not function in the absence of corticosteroids. CONCLUSIONS: Fluvastatin-mediated effects on bone marrow mesenchymal stromal cells were conceivably due to the inhibition of isoprenylation of small guanosin triphosphate binding proteins, occurring for the lack of mevalonate. Altogether these findings suggest that drugs acting on the mevalonate biosynthetic pathway can regulate mesenchymal stromal cell-induced T-cell suppression and B-lymphocyte survival.

Statins enhance formation of phagocyte extracellular traps.

Statins are inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recent clinico-epidemiologic studies correlate patients receiving statin therapy with having reduced mortality associated with severe bacterial infection. Investigating the effect of statins on the innate immune capacity of phagocytic cells against the human pathogen Staphylococcus aureus, we uncovered a beneficial effect of statins on bacterial clearance by phagocytes, although, paradoxically, both phagocytosis and oxidative burst were inhibited. Probing instead for an extracellular mechanism of killing, we found that statins boosted the production of antibacterial DNA-based extracellular traps (ETs) by human and murine neutrophils and also monocytes/macrophages. The effect of statins to induce phagocyte ETs was linked to sterol pathway inhibition. We conclude that a drug therapy taken chronically by millions alters the functional behavior of phagocytic cells, which could have ramifications for susceptibility and response to bacterial infections in these patients.

A multiplexed cell assay in HepG2 cells for the identification of delta-5, delta-6, and delta-9 desaturase and elongase inhibitors.

A multiplexed cell assay has been optimized to measure the activities of fatty acyl-CoA elongase, delta-5 desaturase (Delta5D), delta-6 desaturase (Delta6D), and delta-9 desaturase (Delta9D) together using (14)C-labeled tracers in HepG2 cells, which express the human stearoyl-CoA desaturase-1 isoform (SCD1) exclusively. The Delta5 and Delta9 desaturase activities are indexed by the efficient conversion of [1-(14)C]-eicosatrienoic acid (C20:3, cis-8,11,14) to (14)C-arachidonic acid (C20:4, cis-5,8,11,14) and the conversion of [1-(14)C]-stearic acid to (14)C-oleic acid (C18:1, cis-9), respectively. CP-74006 potently blocks the Delta5D activity with an IC(50) value of 20 nM and simplifies the metabolism of [1-(14)C]-alpha-linolenate (C18:3, cis-9,12,15) by accumulating (14)C-eicosatetraenoic acid (C20:4, cis-8,11,14,17) as the major (14)C-eicosatrienoic acid (C20:3, cis-11,14,17) and (14)C-docosatetraenoic acid (C22:4, cis-10,13,16,19) as the minor metabolites through Delta6 desaturation and elongation. This simplified metabolite spectrum enables the delineation of the Delta6D activity by comparing the combined Delta6D/elongase activity index of the (14)C-(C20:4/C18:3) ratio with the corresponding elongation index of the (14)C-(C20:3/C18:3) ratio following compound treatment. SC-26196 and sterculic acid specifically inhibit the Delta6D and Delta9D activities with an IC(50) value of 0.1 microM and 0.9 microM, respectively. This medium-throughput cell assay provides an efficient tool in the identification of specific desaturase and elongase inhibitors.

Can statin therapy reduce the risk of melanoma? A meta-analysis of randomized controlled trials.

A growing body of literature suggests that statins may have a chemopreventive potential against melanoma through pleiotropic anti-inflammatory, immunomodulatory, and antiangiogenesis mechanisms. Our aim was to examine this association through a detailed meta-analysis of randomized controlled trials (RCTs). A comprehensive search for trials published up to June 2009 was performed, reviews of each study were conducted and data were abstracted. Prior to meta-analysis, the studies were evaluated for publication bias and heterogeneity. Pooled relative risk estimates (RR) and 95% confidence intervals (CIs) were calculated using the fixed- and the random-effects models. Subgroup and sensitivity analyses were also conducted. Sixteen RCTs of statins for cardiovascular outcomes, involving 62,568 individuals with a mean age of 60 years and an average follow-up of nearly 4.7 years, contributed to the analysis. We found no evidence of publication bias (P = 0.47) or heterogeneity among the studies (P = 0.25). Statin use did not significantly affect the risk of developing melanoma assuming either a fixed- (RR = 0.92, 95% CI: 0.67-1.26), or a random-effects model (RR = 0.92, 95% CI: 0.62-1.36). This neutral effect was further supported by the results of subgroup and sensitivity analyses. Our findings do not support a protective effect of statins against melanoma.

Regulation of the brain isoprenoids farnesyl- and geranylgeranylpyrophosphate is altered in male Alzheimer patients.

Post-translational modification of small GTPases by farnesyl- (FPP) and geranylgeranylpyrophosphate (GGPP) has generated much attention due to their potential contribution to cancer, cardiovascular and neurodegenerative diseases. Prenylated proteins have been identified in numerous cell functions and elevated levels of FPP and GGPP have been previously proposed to occur in Alzheimer disease (AD) but have never been quantified. In the present study, we determined if the mevalonate derived compounds FPP and GGPP are increased in brain grey and white matter of male AD patients as compared with control samples. This study demonstrates for the first time that FPP and GGPP levels are significantly elevated in human AD grey and white matter but not cholesterol, indicating a potentially disease-specific targeting of isoprenoid regulation independent of HMG-CoA-reductase. Further suggesting a selective disruption of FPP and GGPP homeostasis in AD, we show that inhibition of HMG-CoA reductase in vivo significantly reduced FPP, GGPP and cholesterol abundance in mice with the largest effect on the isoprenoids. A tentative conclusion is that if indeed regulation of FPP and GGPP is altered in AD brain such changes may stimulate protein prenylation and contribute to AD neuropathophysiology.

HMG-CoA reductase inhibition causes increased necrosis and apoptosis in an in vivo mouse glioblastoma multiforme model.

BACKGROUND: Statins are thought to have tumorolytic properties, reducing angiogenesis by inhibiting pro-angiogenic factors and inducing apoptosis of mural pericytes within the tumor vascular tree. MATERIALS AND METHODS: An orthotopic mouse glioblastoma (GL-26) model was used to investigate the effect of simvastatin on glioblastoma vasculature in vivo. GL-26 cells were implanted into the striatum of C5LKa mice treated with either control, low- or high-dose simvastatin. Brains were analyzed for necrotic volume, apoptosis, morphology and pericytic cells within the vascular tree. RESULTS: Low-dose simvastatin increased necrosis and apoptosis compared to both control and high-dose simvastatin groups. High-dose simvastatin increased vessel caliber by reducing pericytic cells along the tumor vessel wall compared to both control and low-dose simvastatin groups. CONCLUSION: Simvastatin has a dual effect on tumorigenesis. At high doses, it may worsen instead of 'normalizing' tumor angio-architecture, albeit low doses affect tumor cell survival by promoting necrosis and apoptosis.

Statins inhibit toll-like receptor 4-mediated lipopolysaccharide signaling and cytokine expression.

OBJECTIVE: Toll-like receptor 4 (TLR4) is the main receptor for Lipopolysaccharide (LPS). Two relatively common variants of the TLR4 gene are present, resulting in changes from aspartic acid (D) to glycine (G) at residue 299 and from threonine (T) to isoleucine (I) at residue 399, respectively. It has been shown that statins have a greater effect on lowering risk of cardiovascular events in individuals carrying the 299G allele than in those not carrying this allele. We investigated possible mechanisms underlying this synergy of statin treatment and TLR4 genotype. METHODS AND RESULTS: In cells expressing the 299D-399T TLR4, LPS activated the transcription factor NFkappaB and increased the expression of interleukin-6 and tumor necrosis factor-alpha, and these effects were reduced by pretreatment of the cells with pravastatin or simvastatin. LPS-induced NFkappaB activation and interleukin-6 and tumor necrosis factor-alpha expression were substantially reduced in cell expressing the 299G-399T or 299D-399I variant, and undetectable in cells expressing the 299G-399I TLR4. The 3-hydroxy-3-methylglutaryl coenzyme A pathway inhibitors, Y27632 and GGTI-286, exhibited a similar effect to statins, suggesting that the inhibitory effect of statins was mediated by the 3-hydroxy-3-methylglutaryl coenzyme A pathway. CONCLUSION: The results of this study indicate that the TLR4 variations and statins have an additive inhibitory effect on TLR4-mediated inflammatory response, providing a potential explanation for the finding that the beneficial effect of statins on cardiovascular risk is dependent on TLR4 genotype.