Decreased Cholesterol Biosynthesis Research Articles

Multiomics Profiling Distinguishes Sebaceous Carcinoma from Benign Sebaceous Neoplasms and Provides Insight into the Genetic Evolution of Sebaceous Carcinogenesis.

Sebaceous carcinoma is the third most common nonkeratinocyte skin cancer in the United States with 1,000 cases per year. The clinicopathologic features of sebaceous carcinoma and benign sebaceous neoplasms (adenomas, sebaceomas) can overlap, highlighting the need for molecular biomarkers to improve classification. This study describes the genomic and transcriptomic landscape of sebaceous neoplasms in order to understand tumor etiology and biomarkers relevant for diagnosis and treatment. We performed whole-genome sequencing (WGS) and whole-transcriptome sequencing (WTS) of sebaceous neoplasms from six academic and two federal healthcare facilities in the United States diagnosed between January 1, 1999, and December 31, 2021. We evaluated 98 sebaceous neoplasms: 64 tumors (32 adenomas, 2 sebaceomas, 5 atypical sebaceous neoplasms, 25 carcinomas) had sufficient material for WGS, 96 tumors (42 adenomas, 11 sebaceomas, 8 atypical sebaceous neoplasms, 35 carcinomas) had sufficient material for WTS, and 62 tumors (31 adenomas, 2 sebaceomas, 5 atypical sebaceous neoplasms, 24 carcinomas) had sufficient material for combined WGS and WTS. Overall, we found decreased cholesterol biosynthesis and increased TP53 mutations, copy number gains (chromosome 6, 8q, and/or 18), and tumor mutation burden-high (>10 mutations/MB) in carcinomas compared to adenomas. Although diminished compared to adenomas, most carcinomas still had higher cholesterol biosynthesis than nonmalignant skin. Multiomics profiling also supported a precancerous model of tumor evolution with sebaceomas and atypical sebaceous neoplasms being likely intermediate lesions. The study findings highlight key diagnostic biomarkers for sebaceous carcinoma and suggest that immunotherapy and modulation of cholesterol biosynthesis could be effective treatment strategies.

Pharmacological modulation of lipid metabolism in a human cell line alters protein content and signalling of secreted extracellular vesicles

Background and Aims : Extracellular Vesicles (EVs) are an attractive pharmacological target due to their involvement in cell-cell communications. From a lipid point of view, EVs are composed by phospholipids and cholesterol. The latter, together with the phospholipid bis-monoacylglycerophosphate (BMP), plays a pivotal role in EVs fate, since their balance drives late endosomes towards secretion or lysosomal recycling. Based on these premises, we decreased cholesterol biosynthesis with the HMG-CoA reductase inhibitor simvastatin and BMP degradation by KT182 in a melanoma cell line and we evaluated the effects on secreted EVs and their potential functional alterations.

Combination treatment of docetaxel with caffeic acid phenethyl ester suppresses the survival and the proliferation of docetaxel-resistant prostate cancer cells via induction of apoptosis and metabolism interference

BackgroundDocetaxel has been approved by USFDA as a first-line treatment for castration-resistant prostate cancer (CRPC) patients. Patients receiving androgen deprivation therapy along with docetaxel result in superior survival, lower serum prostate specific antigen (PSA) level, and better quality of life. However, a significant proportion of these patients ultimately develop resistance to docetaxel within months. Caffeic acid phenethyl ester (CAPE), one of the main bioactive components extracted from the propolis, has been reported to be effective for repressing the tumor growth, the migration and invasion of prostate cancer (PCa) cells, as well as the downstream signaling and stability of androgen receptor (AR). We hence determined if combination treatment of docetaxel with CAPE can suppress the proliferation and the survival of docetaxel-resistant PCa cells.MethodsWe established docetaxel-resistant PC/DX25 and DU/DX50 CRPC cell lines from PC-3 and DU-145 human PCa cells, respectively. Proliferation assay, MTT assay, flow cytometry with Annexin V staining, Comet Assay, and nude mice xenograft model were applied to determine the effects of combination treatment on cell proliferation and survival of the docetaxel-resistant PCa cells. Micro-Western Array (MWA) and qRT-PCR were used to investigate the molecular mechanism lying underneath.ResultsCombination treatment effectively suppressed the proliferation, survival and tumor growth of docetaxel-resistant PCa cells both in vitro and in nude mice. Comet assay and flow cytometry indicated that combination treatment induced apoptosis in docetaxel-resistant PCa cells. MWA and Western blotting assay revealed that combination treatment suppressed protein expression of Bcl-2, AKT2, c-Myc, apoptosis and caspase activation inhibitor (AVEN), pyruvate kinase M2 (PKM2) but increased protein expression of Bax, caspase 3, cytochrome c, glucose-6-phosphate dehydrogenase (G6PD) and acylglycerol kinase (AGK). Overexpression of Bcl-2 in the docetaxel-resistant PCa cells enhanced cell proliferation of docetaxel-resistant PCa cells under combination treatment. Analysis with qRT-PCR suggested that combination treatment decreased cholesterol biosynthesis genes DHCR24 (24-dehydrocholesterol reductase) and LSS (lanosterol synthase) but increased genes involved in glycolysis and TCA cycle.ConclusionsCombination treatment of docetaxel with CAPE effectively suppressed the proliferation and survival of docetaxel-resistant PCa cells via inhibition of Bcl-2 and c-Myc as well as induction of metabolism interference. Combination treatment can be beneficial for patients with docetaxel-resistant PCa.

Peroxisomal β-oxidation stimulates cholesterol biosynthesis in the liver in diabetic mice

Although diabetes normally causes an elevation of cholesterol biosynthesis and induces hypercholesterolemia in animals and human, the mechanism linking diabetes to the dysregulation of cholesterol biosynthesis in the liver is not fully understood. As liver peroxisomal β-oxidation is induced in the diabetic state and peroxisomal oxidation of fatty acids generates free acetate, we hypothesized that peroxisomal β-oxidation might play a role in liver cholesterol biosynthesis in diabetes. Here, we used erucic acid, a specific substrate for peroxisomal β-oxidation, and 10,12-tricosadiynoic acid, a specific inhibitor for peroxisomal β-oxidation, to specifically induce and suppress peroxisomal β-oxidation. Our results suggested that induction of peroxisomal β-oxidation increased liver cholesterol biosynthesis in streptozotocin-induced diabetic mice. We found that excessive oxidation of fatty acids by peroxisomes generated considerable free acetate in the liver, which was used as a precursor for cholesterol biosynthesis. In addition, we show that specific inhibition of peroxisomal β-oxidation decreased cholesterol biosynthesis by reducing acetate formation in the liver in diabetic mice, demonstrating a crosstalk between peroxisomal β-oxidation and cholesterol biosynthesis. Based on these results, we propose that induction of peroxisomal β-oxidation serves as a mechanism for a fatty acid-induced upregulation in cholesterol biosynthesis and also plays a role in diabetes-induced hypercholesterolemia.

Maprotiline Suppresses Cholesterol Biosynthesis and Hepatocellular Carcinoma Progression Through Direct Targeting of CRABP1.

Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related death and has a poor prognosis worldwide, thus, more effective drugs are urgently needed. In this article, a small molecule drug library composed of 1,056 approved medicines from the FDA was used to screen for anticancer drugs. The tetracyclic compound maprotiline, a highly selective noradrenergic reuptake blocker, has strong antidepressant efficacy. However, the anticancer effect of maprotiline remains unclear. Here, we investigated the anticancer potential of maprotiline in the HCC cell lines Huh7 and HepG2. We found that maprotiline not only significantly restrained cell proliferation, colony formation and metastasis in vitro but also exerted antitumor effects in vivo. In addition to the antitumor effect alone, maprotiline could also enhance the sensitivity of HCC cells to sorafenib. The depth studies revealed that maprotiline substantially decreased the phosphorylation of sterol regulatory element-binding protein 2 (SREBP2) through the ERK signaling pathway, which resulted in decreased cholesterol biosynthesis and eventually impeded HCC cell growth. Furthermore, we identified cellular retinoic acid binding protein 1 (CRABP1) as a direct target of maprotiline. In conclusion, our study provided the first evidence showing that maprotiline could attenuate cholesterol biosynthesis to inhibit the proliferation and metastasis of HCC cells through the ERK-SREBP2 signaling pathway by directly binding to CRABP1, which supports the strategy of repurposing maprotiline in the treatment of HCC.

Antiobesity and hypolipidemic effects of protease A-digested crude-chalaza hydrolysates in a high-fat diet

• Protease A-digested crude chalaza hydrolysates (CCH-As) own antiobesity effects. • CCH-As reduce liver/perirenal-adipose-tissue sizes in high-fat diet fed hamsters. • CCH-As improve serum lipids and antioxidant levels in high-fat diet fed hamsters. • The antiobesity effect of CCH-As is due to enhancing lipolysis of adipose tissues. • CCH-As downregulate cholesterol biosynthesis and upregulate cholesterol catabolism. A high-fat diet is a major risk factor for obesity and heart failure. Our previous study has demonstrated that protease A-digested crude-chalaza hydrolysates (CCH-As) with a specific free amino-acid and carnosine/anserine profile show lipid-lowering and antioxidant activities against chronic alcohol consumption. The antiobesity and hypolipidemic effects of our CCH-As against a high-fat diet was to investigated in this study. In high-fat-diet fed hamsters, supplementing CCH-As reduced liver/perirenal-adipose-tissue sizes, and serum triglyceride, LDLC/HDLC, and TBARS values (p < 0.05). Supplementing CCH-As increased lipolysis (hormone sensitive lipase, carnitine palmitoyltransferase 1, and uncoupling protein 2) in the perirenal-adipose tissues of high-fat-diet fed hamsters (p < 0.05); meanwhile decreased cholesterol biosynthesis (squalene synthase), and upregulated bile-acid biosynthesis (cholesterol 7 alpha-hydroxylase) and cholesterol clearance ability (low-density lipoprotein receptor) in livers (p < 0.05). Daily fecal lipid and bile-acid outputs were also increased in high-fat-diet fed hamsters cotreated with CCH-As. Therefore, this CCH-A can be characterized as an antiobesity and cardioprotective ingredient.

The Interplay between Retinal Pathways of Cholesterol Output and Its Effects on Mouse Retina.

In mammalian retina, cholesterol excess is mainly metabolized to oxysterols by cytochromes P450 27A1 (CYP27A1) and 46A1 (CYP46A1) or removed on lipoprotein particles containing apolipoprotein E (APOE). In contrast, esterification by sterol-O-acyltransferase 1 (SOAT) plays only a minor role in this process. Accordingly, retinal cholesterol levels are unchanged in Soat1−/− mice but are increased in Cyp27a1−/−Cyp46a1−/− and Apoe−/− mice. Herein, we characterized Cyp27a1−/−Cyp46a1−/−Soat1−/− and Cyp27a1−/−Cyp46a1−/−Apoe−/− mice. In the former, retinal cholesterol levels, anatomical gross structure, and vasculature were normal, yet the electroretinographic responses were impaired. Conversely, in Cyp27a1−/−Cyp46a1−/−Apoe−/− mice, retinal cholesterol levels were increased while anatomical structure and vasculature were unaffected with only male mice showing a decrease in electroretinographic responses. Sterol profiling, qRT-PCR, proteomics, and transmission electron microscopy mapped potential compensatory mechanisms in the Cyp27a1−/−Cyp46a1−/−Soat1−/− and Cyp27a1−/−Cyp46a1−/−Apoe−/− retina. These included decreased cholesterol biosynthesis along with enhanced formation of intra- and extracellular vesicles, possibly a reserve mechanism for lowering retinal cholesterol. In addition, there was altered abundance of proteins in Cyp27a1−/−Cyp46a1−/−Soat1−/− mice that can affect photoreceptor function, survival, and retinal energy homeostasis (glucose and fatty acid metabolism). Therefore, the levels of retinal cholesterol do not seem to predict retinal abnormalities, and it is rather the network of compensatory mechanisms that appears to determine retinal phenotype.

Whole Grains in Amelioration of Metabolic Derangements.

Daily diet influences whole body metabolism, and intricately linked to the prevention or progression of metabolic diseases including obesity, diabetes and cardiovascular diseases. Several epidemiological and large scale studies have shown that diets enriched with whole grains improves metabolic function and protect from the development of metabolic diseases. Direct impact of whole grain diet can be mediated on several levels of metabolic functions i.e. reduced glycemic index, improved fat oxidation potential, increased cholesterol clearance or decreased cholesterol biosynthesis and modulation of gut microbiome. In this article we reviewed several studies indicating the beneficial effects of whole grain diets on metabolic functions, as well as discussed the potential active phytochemicals present in these whole grain foods to contribute in modulation of metabolic function in our body.

Aqueous or lipid components of atherosclerotic lesion increase macrophage oxidation and lipid accumulation

Introduction and objectiveUnderstanding the interactions among atherosclerotic plaque components and arterial macrophages, is essential for elucidating the mechanisms involved in the development of atherosclerosis. We assessed the effects of lesion extracts on macrophages. MethodsMouse peritoneal macrophages from atherosclerotic normoglycemic or hyperglycemic apoE−/− mice were incubated with aortic aqueous or with aortic lipidic extracts (mAAE or mALE) derived from these mice. In parallel, J774A.1 cultured macrophages were incubated with increasing concentrations of extracts prepared from human carotid lesions: polar lesion aqueous extract (hLAE), nonpolar lesion lipid extract (hLLE), or with their combination. In all the above systems we performed analyses of macrophage oxidative status, cholesterol, and triglyceride metabolism. ResultsAqueous or lipid extracts from either mice aorta or from human carotid lesions significantly increased macrophage oxidative stress as determined by reactive oxygen species (ROS) analysis. In parallel, a compensatory increase in the cellular antioxidant paraoxonase2 (PON2) activity and in macrophage glutathione content were observed following incubation with all extracts. Macrophage triglyceride mass and triglyceride biosynthesis rate were both significantly increased following treatment with the lipid extracts, secondary to upregulation of DGAT1. All extracts decreased cholesterol biosynthesis rate, through downregulation of HMGCR, the rate limiting enzyme in cholesterol biosynthesis. The combination of the human lesion extracts had the most significant effects. ConclusionThe present study demonstrates that atherosclerotic plaque constituents enhance macrophage cellular oxidative stress, and accumulation of cholesterol and triglycerides, as shown in both in vivo and in vitro model systems.

How Low Cholesterol Is Good for Anti-viral Immunity

York etal. identify a role for decreased cholesterol biosynthesis in virally infected cells as a critical event in the induction of the anti-viral response. The mechanism involves enhanced signaling by STING in the ER membrane in response to the second messenger cGAMP, promoting increased transcription of type I interferons. The authors suggest that a "lipid code" is being sensed by STING to allow it to signal.

Human carotid atherosclerotic lesion protein components decrease cholesterol biosynthesis rate in macrophages through 3-hydroxy-3-methylglutaryl-CoA reductase regulation.

Atherosclerosis is characterized by the formation of cholesterol-loaded macrophages, which are turned into foam cells, the hallmark of early atherogenesis. As part of ongoing research on the interactions among human carotid lesion components and blood elements, the effect of plaque homogenate on macrophage cholesterol biosynthesis rate was examined. Human carotid plaques were ground, extracted with phosphate-buffered saline (homogenate), and then added to the macrophage medium. This extract decreased macrophage cholesterol biosynthesis rate up to 50% in a dose-dependent manner. Cholesterol or lipoproteins were separated from the homogenate and added to the MQ medium. Unlike the homogenate, neither free cholesterol nor the lipoproteins were able to inhibit cholesterol biosynthesis rate under the above experimental concentration, suggesting that the homogenate-induced cholesterol biosynthesis inhibition in our experimental system was not owing to the feedback inhibition of cholesterol. Furthermore, the homogenate remaining after lipoprotein removal (lipoprotein-deficient homogenate) also decreased cholesterol biosynthesis rate, whereas boiled homogenate or phospholipids extracted from the homogenate decreased macrophage cholesterol biosynthesis rate only partially. Finally, cholesterol biosynthesis inhibition was achieved only upon using the precursor [(3)H]acetate, but not [(14)C]mevalonate, suggesting that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA Reductase), the rate-limiting enzyme in the cholesterol biosynthesis pathway, is involved in the above antiatherogenic effect of the homogenate, whereas the treatment with homogenate decreased HMGCoA Reductase mRNA. Proteins and phospholipids from human carotid lesion homogenate decrease cholesterol biosynthesis rate in macrophages secondary to HMGCoA Reductase feedback regulation. Such an effect may delay foam cell formation and atherosclerosis progression.

FoxO4 interacts with the sterol regulatory factor SREBP2 and the hypoxia inducible factor HIF2α at the CYP51 promoter

The late steps of cholesterol biosynthesis are oxygen demanding, requiring eleven oxygen molecules per synthesized cholesterol molecule. A key enzymatic reaction, which occurs at the top of the Bloch and Kandutsch-Russell pathways, is the demethylation of lanosterol and dihydrolanosterol (DHL). This reaction is catalyzed by lanosterol 14α demethylase (CYP51) and requires three oxygen molecules. Thus, it is the first step in the distal pathway to be susceptible to oxygen deprivation. Having previously identified that the forkhead transcription factor 4 (FoxO4) represses CYP51 expression, we aimed to characterize its role at the CYP51 promoter. Hypoxia-treated 3T3L1 cells showed decreased cholesterol biosynthesis, accumulation of lanosterol/DHL, and stimulation of FoxO4 expression and its cytoplasmic translocation to the nucleus. Transfection assays with a CYP51 promoter reporter gene revealed that FoxO4 and sterol regulatory element binding protein (SREBP)2 exert a stimulatory effect, whereas FoxO4 and the hypoxia inducible factor (HIF)2α repress CYP51 promoter activity. Electromobility shift, chromatin immunoprecipitation, pull-down, and coimmunoprecipitation assays show that FoxO4 interacts with SREBP2 and HIF2α to modulate CYP51 promoter activity. We also show an inverse correlation between FoxO4 and CYP51 in adipose tissue of ob/ob mice and mouse fetal cortical neurons exposed to hypoxia. Overall, these studies demonstrate a role for FoxO4 in the regulation of CYP51 expression.

Gamma-Tocotrienol and Simvastatin Synergistically Induce Cytotoxicity In Leukemia Cell Lines, K-562 and HL-60

IntroductionStatins and tocotrienols modulate the cholesterol biosynthesis pathway by inhibiting the 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase. Tocotrienols modulate HMG-CoA reductase by post-transcriptional downregulation. In addition, tocotrienols contain a farnesol moiety in its side-chain that triggers degradation of HMG-CoA reductase. These effects lead to suppression of cell proliferation, cell cycle arrest, and apoptosis. Several studies have shown that statins have suppressive effects in in vitro experiments on acute myelocytic leukemia cell lines. Since both statins and gamma-tocotrienol are associated with decreased cholesterol biosynthesis, we hypothesized that if the cytotoxicity of these drugs on cancer cells is related to impaired biosynthesis of cholesterol, combination of them could synergize in cytotoxicity on leukemic cells. Materials and MethodsK-562 and HL-60 leukemia cells were grown in Iscove's Modified Dulbecco's medium with penicillin/streptomycin, 10% fetal bovine serum and 20% fetal bovine serum added respectively. K-562 and HL-60 leukemia cells were seeded in 96 well plates, grown overnight, and treated for 24, 48 and 72 hours with simvastatin in concentrations of 1,2,4, and5 µM; gamma-tocotrienol in concentrations of 20, 40, and 80 µM; and a combination of the two drugs in the same concentrations. For 24 hour dose, cells were seeded at a density of 5000/well and for 48 and 72 hour doses, at 3500/well. Following the treatment, MTS/PMS reagent (Promega, Madison, WI), [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] mixed with and an electron coupling reagent (phenazine methosulfate), was added at 40 µg/well and incubated at 37°C for 2 hours. We measured the solubilized formazan crystals at 450 nm as an indicator of cytotoxicity. The presence of ATP as an indicator of cell viability was measured with the CellTiter Glo® assay (Promega). Cells were again seeded, grown overnight, and dosed as indicated above. Following treatment, the assay was conducted as specified by the manufacturer. ResultsBoth simvastatin and gamma-tocotrienol induce cytotoxicity in K-562 and HL-60 cell lines by the MTS and Cell Titer Glo Assays. The IC50 at 72 hour incubation are as follows 1) HL-60: simvastatin - 16.543 uM, gamma tocotrienol - 36.297 uM; 2) K-562: simvastatin - 5.235 uM, gamma tocotrienol - 34.947 uM. We used CompuSyn to calculate the IC50. When combined, simvastatin and gamma-tocotrienol exhibit synergy at lower concentrations when examined by isobologram analysis. [Display omitted] [Display omitted] ConclusionGamma-tocotrienol, an isoform of vitamin E, and simvastatin, a cholesterol lowering drug exhibit synergy in induction of cytotoxicity in K-562 and HL-60 leukemia cell lines. Rescue experiments and mechanistic pathway analysis are being explored to confirm these observations. Disclosures:No relevant conflicts of interest to declare.

Abstract 23: miR-455-3p Overrides Transcription and Inhibits Cholesterol Biosynthesis

Cholesterol biosynthesis is under tight control by sterol sensing proteins and transcriptional regulation; however, recent evidence suggests that this pathway may also be post-transcriptionally regulated by microRNAs (miRNA). miR-455-3p was found to be significantly up-regulated in mouse livers with high-fat diets, which coincided with significant repression of miR-455-3p target genes, as determined by cumulative distribution fraction analysis of gene expression microarray data. Ten of fourteen cholesterol biosynthetic enzymes were also found to be significantly down-regulated in the livers of these mice; however, sterol regulatory element binding transcription factor 2 (SREBF2) activity was found to be significantly increased, not decreased. Multiple enzymes in the cholesterol biosynthetic pathway were predicted to be miR-455-3p targets; therefore, we hypothesized that miR-455-3p is a master regulator of cholesterol production through direct and indirect targeting of enzymes in the biosynthetic pathway. Furthermore, inhibition of miR-455-3p with locked-nucleic acids (LNA) significantly increased mRNA levels of all cholesterol enzymes analyzed in vitro, including HMGCR, CYP51A1, DHCR7, SC4MOL, and SC5DL. Conversely, miR-455-3p over-expression resulted in decreased mRNA levels of specific cholesterol biosynthesis enzymes. Most importantly, miR-455-3p inhibition significantly increased cholesterol biosynthesis in multiple cell lines from multiple species, as determined by radiolabeled acetate incorporation assays; whereas miR-455-3p transfection decreased cholesterol biosynthesis. Using gene reporter (luciferase) assays, miR-455-3p was found to directly target the 3’ untranslated region of 7-dehydrocholesterol reductase (DHCR7). Inhibition of miR-455-3p in DHCR7 mutated cell lines representing Smith-Lemli Opitz Syndrome, which results from non-complete dysfunction of DHCR7, significantly increased DHCR7 mRNA levels and cholesterol biosynthesis. In summary, miR-455-3p is a powerful inhibitor of cholesterol biosynthesis through post-transcriptional regulation that likely overrides SREBF2 transcriptional regulation within cholesterol metabolism.

Cholesteryl ester accumulation and accelerated cholesterol absorption in intestine-specific hormone sensitive lipase-null mice

Hormone sensitive lipase (HSL) regulates the hydrolysis of acylglycerols and cholesteryl esters (CE) in various cells and organs, including enterocytes of the small intestine. The physiological role of this enzyme in enterocytes, however, stayed elusive. In the present study we generated mice lacking HSL exclusively in the small intestine (HSLiKO) to investigate the impact of HSL deficiency on intestinal lipid metabolism and the consequences on whole body lipid homeostasis. Chow diet-fed HSLiKO mice showed unchanged plasma lipid concentrations. In addition, feeding with high fat/high cholesterol (HF/HC) diet led to unaltered triglyceride but increased plasma cholesterol concentrations and CE accumulation in the small intestine. The same effect was observed after an acute cholesterol load. Gavaging of radioactively labeled cholesterol resulted in increased abundance of radioactivity in plasma, liver and small intestine of HSLiKO mice 4h post-gavaging. However, cholesterol absorption determined by the fecal dual-isotope ratio method revealed no significant difference, suggesting that HSLiKO mice take up the same amount of cholesterol but in an accelerated manner. mRNA expression levels of genes involved in intestinal cholesterol transport and esterification were unchanged but we observed downregulation of HMG-CoA reductase and synthase and consequently less intestinal cholesterol biosynthesis. Taken together our study demonstrates that the lack of intestinal HSL leads to CE accumulation in the small intestine, accelerated cholesterol absorption and decreased cholesterol biosynthesis, indicating that HSL plays an important role in intestinal cholesterol homeostasis.

Simvastatin Downregulates HER2 via Upregulation of PEA3 to Induce Cell Death in HER2-Positive Breast Cancer Cells

Simvastatin is a widely used cholesterol-adjusting drug that selectively inhibits the 3-hyrdoxy-3-methylglutaryl-coenzyme A reductase, leading to decreased cholesterol biosynthesis. Notably, through this activity, simvastatin exerts antiproliferative and proapoptotic effects on various cancer cells, including non-small cell lung and breast cancer. Although statin-induced breast cancer cell death is nitric oxide inducible and arginase dependent, we report alternative mechanisms relative to the antitumor function of simvastatin in breast cancer cells. Simvastatin induced cell death in MDA-MB-361, SK-Ov3, and SKBR3, HER2-overexpressing cell lines, in both time- and dose-dependent manners, but did not exert cytotoxicity in MCF10A and MDA-MB-231, HER2 low/negative cell lines. The protein expression of HER2 decreased after the cells were treated with simvastatin; however, HER2 protein and mRNA stabilities were not changed. Furthermore, simvastatin inhibited the activity of the HER2 promoter. Simvastatin-induced cytotoxicity and promoter activity repression were reversed by mevalonate and GGPP, the immediate metabolic products of the acetyl CoA/3-hyrdoxy-3-methylglutaryl CoA reductase reaction and the isoprenoid of the mevalonate cascade, respectively. In addition, simvastatin treatment induced the expression of PEA3, which is a HER2 promoter inhibitor. The use of siRNA to downregulate expression of PEA3 inhibited the simvastin-induced HER2 repression and cell death. These findings provide alternative mechanisms for the antitumor effects of simvastatin, suggesting that simvastatin could also be used as a combination therapy with other chemotherapy agents in HER2-positive patients.

Fluorescence image screening for chemical compounds modifying cholesterol metabolism and distribution

An automated fluorescence microscopy assay using a nontoxic cholesterol binding protein, toxin domain 4, (D4), was developed in order to identify chemical compounds modifying intracellular cholesterol metabolism and distribution. Using this method, we screened a library of 1,056 compounds and identified 35 compounds that decreased D4 binding to the cell surface. Among them, 8 compounds were already reported to alter the biosynthesis or the intracellular distribution of cholesterol. The remaining 27 hit compounds were further analyzed biochemically and histochemically. Cell staining with another fluorescent cholesterol probe, filipin, revealed that 17 compounds accumulated cholesterol in the late endosomes. Five compounds decreased cholesterol biosynthesis, and two compounds inhibited the binding of D4 to the membrane. This visual screening method, based on the cholesterol-specific probe D4 in combination with biochemical analyses, is a cell-based, sensitive technique for identifying new chemical compounds and modifying cholesterol distribution and metabolism. Furthermore, it is suitable for high-throughput analysis for drug discovery.

Statin-Induced Breast Cancer Cell Death: Role of Inducible Nitric Oxide and Arginase-Dependent Pathways

Statins are widely used cholesterol-lowering drugs that selectively inhibit the enzyme 3-hydroxy-3-methylglutaryl CoA reductase, leading to decreased cholesterol biosynthesis. Emerging data indicate that statins stimulate apoptotic cell death in several types of proliferating tumor cells, including breast cancer cells, which is independent of its cholesterol-lowering property. The objective here was to elucidate the molecular mechanism(s) by which statins induce breast cancer cell death. Fluvastatin and simvastatin (5-10 mumol/L) treatment enhanced the caspase-3-like activity and DNA fragmentation in MCF-7 cells, and significantly inhibited the proliferation of MCF-7 cells but not MCF-10 cells (noncancerous epithelial cells). Statin-induced cytotoxic effects were reversed by mevalonate, an immediate metabolic product of the acetyl CoA/3-hydroxy-3-methylglutaryl CoA reductase reaction. Both simvastatin and fluvastatin enhanced nitric oxide ((.)NO) levels which were inhibited by mevalonate. Statin-induced (.)NO and tumor cell cytotoxicity were inhibited by 1400W, a more specific inhibitor of inducible nitric oxide synthase (iNOS or NOS II). Both fluvastatin and simvastatin increased iNOS mRNA and protein expression. Stimulation of iNOS by statins via inhibition of geranylgeranylation by GGTI-298, but not via inhibition of farnesylation by FTI-277, enhanced the proapoptotic effects of statins in MCF-7 cells. Statin-mediated antiproliferative and proapoptotic effects were exacerbated by sepiapterin, a precursor of tetrahydrobiopterin, an essential cofactor of (.)NO biosynthesis by NOS. We conclude that iNOS-mediated (.)NO is responsible in part for the proapoptotic, tumoricidal, and antiproliferative effects of statins in MCF-7 cells.

Selective Up-regulation of LXR-regulated Genes ABCA1, ABCG1, and APOE in Macrophages through Increased Endogenous Synthesis of 24(S),25-Epoxycholesterol

Liver X receptor (LXR) activation represents a mechanism to prevent macrophage foam cell formation. Previously, we demonstrated that partial inhibition of oxidosqualene:lanosterol cyclase (OSC) stimulated synthesis of the LXR agonist 24(S),25-epoxycholesterol (24(S),25-epoxy) and enhanced ABCA1-mediated cholesterol efflux. In contrast to a synthetic, nonsteroidal LXR activator, TO-901317, triglyceride accumulation was not observed. In the present study, we determined whether endogenous 24(S),25-epoxy synthesis selectively enhanced expression of macrophage LXR-regulated cholesterol efflux genes but not genes that regulate fatty acid metabolism. THP-1 human macrophages incubated with the OSC inhibitor (OSCi) RO0714565 (15 nM) significantly reduced cholesterol synthesis and maximized synthesis of 24(S),25-epoxy. Endogenous 24(S),25-epoxy increased ABCA1, ABCG1, and APOE mRNA abundance and consequently increased cholesterol efflux to apoAI. In contrast, OSCi had no effect on LXR-regulated genes LPL (lipoprotein lipase) and FAS (fatty acid synthase). TO-901317 (>or=10 nM) significantly enhanced expression of all genes examined. OSCi and TO-901317 increased the mRNA and precursor form of SREBP-1c, a major regulator of fatty acid and triglyceride synthesis. However, conversion of the precursor to the active form (nSREBP-1c) was blocked by OSCi-induced 24(S),25-epoxy but not by TO-901317 (>or=10 nm), which instead markedly increased nSREBP-1c. Disruption of nSREBP-1c formation by 24(S),25-epoxy accounted for diminished FAS and LPL expression. In summary, endogenous synthesis of 24(S),25-epoxy selectively up-regulates expression of macrophage LXR-regulated cholesterol efflux genes without stimulating genes linked to fatty acid and triglyceride synthesis.

“New” hepatic fat activates PPARα to maintain glucose, lipid, and cholesterol homeostasis

De novo lipogenesis is an energy-expensive process whose role in adult mammals is poorly understood. We generated mice with liver-specific inactivation of fatty-acid synthase (FAS), a key lipogenic enzyme. On a zero-fat diet, FASKOL (FAS knockout in liver) mice developed hypoglycemia and fatty liver, which were reversed with dietary fat. These phenotypes were also observed after prolonged fasting, similarly to fasted PPARalpha-deficiency mice. Hypoglycemia, fatty liver, and defects in expression of PPARalpha target genes in FASKOL mice were corrected with a PPARalpha agonist. On either zero-fat or chow diet, FASKOL mice had low serum and hepatic cholesterol levels with elevated SREBP-2, decreased HMG-CoA reductase expression, and decreased cholesterol biosynthesis; these were also corrected with a PPARalpha agonist. These results suggest that products of the FAS reaction regulate glucose, lipid, and cholesterol metabolism by serving as endogenous activators of distinct physiological pools of PPARalpha in adult liver.