Importance of cholesterol and oxysterols metabolism in the pharmacology of tamoxifen and other AEBS ligands

Tamoxifen is one of the major drugs used for the hormonotherapy of estrogen receptor positive breast cancers. However, its therapeutic efficacy can be limited by acquired resistance and tumor recurrence can occur after several years of treatment. Tamoxifen is known as the prototypical modulator of estrogen receptors, but other targets have been identified that could account for its pharmacology. In particular, tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS) and inhibits cholesterol esterification at therapeutic doses. We have recently shown that the AEBS was a hetero-oligomeric complex composed of 3β-hydroxysterol-Δ(8)-Δ(7)-isomerase and 3β-hydroxysterol-Δ(7)-reductase, that binds different structural classes of ligands, including selective estrogen receptor modulators, several sigma receptor ligands, poly-unsaturated fatty acids and ring B oxysterols. We established a link between the modulation of cholesterol metabolism by tamoxifen and other AEBS ligands and their capacity to induce breast cancer cell differentiation, apoptosis and autophagy. Moreover, we showed that the AEBS carries out cholesterol-5,6-epoxide hydrolase activity and established that cholesterol-5,6-epoxide hydrolase is a new target for tamoxifen and other AEBS ligands. Finally in this review, we report on recent data from the literature showing how the modulation of cholesterol and oxysterol metabolism can be linked to the antitumor and chemopreventive properties of tamoxifen, and give new perspectives to improve the clinical outcome of the hormonotherapy of breast cancers.     

Dehydroepiandrosterone fatty acyl esters in high density lipoprotein: interaction with human vascular endothelial cells and vascular responses ex vivo

Dehydroepiandrosterone (DHEA) fatty acyl esters once incorporated in high density lipoprotein (HDL) induce a stronger vasodilatory response in rat mesenteric arteries ex vivo compared to native HDL. We studied the role of HDL receptor, scavenger receptor class B, type 1 (SR-B1), as well as estrogen and androgen receptors in the vasodilatory response of HDL-associated DHEA fatty acyl esters. Using cultured human vascular endothelial cells (HUVEC), we investigated the possible internalization and cellular response of HDL-associated DHEA esters. We prepared DHEA ester-enriched HDL by incubating human plasma in the presence of DHEA. After isolation and purification, HDL was added in cumulative doses to arterial rings precontracted with noradrenaline. Inhibition of the function of SR-B1 almost completely abolished maximal vasorelaxation by DHEA-enriched HDL while estrogen or androgen receptor blockage had no significant effect. When HUVECs were incubated in the presence of [³H]DHEA ester-enriched HDL, the amount of intracellular [³H]-radioactivity increased steadily during 24 h. Blocking of SR-B1 reduced this uptake by a mean of 30%. The proportion of unesterified [³H]DHEA, as analyzed by thin-layer chromatography, increased intracellularly and in the cell culture media after several hours of incubation of the cells in the presence of [³H]DHEA ester-enriched HDL. This indicated slow hydrolysis of DHEA fatty acyl esters and subsequent excretion of unesterified DHEA by the cells. In conclusion, DHEA-enriched HDL induced vasorelaxation via the SR-B1-facilitated pathway. However, this vasodilation is not likely to be attributed to rapid hydrolysis of HDL-associated DHEA esters by the vascular endothelium.     

A quantitative model for metabolic intervention using gut microbes

As medicine shifts toward precision-based and personalized therapeutics, utilizing more complex biomolecules to treat increasingly difficult and rare conditions, microorganisms provide an avenue for realizing the production and processing necessary for novel drug pipelines. More so, probiotic microbes can be co-opted to deliver therapeutics by oral administration as living drugs, able to survive and safely transit the digestive tract. As living therapeutics are in their nascency, traditional pharmacokinetic–pharmacodynamic (PK–PD) models for evaluating drug candidates are not appropriate for this novel platform. Using a living therapeutic in late-stage clinical development for phenylketonuria (PKU) as a case study, we adapt traditional oral drug delivery models to properly evaluate and inform the engineering of living therapeutics. We develop the adapted for living therapeutics compartmental absorption and transit (ALT-CAT) model to provide metrics for drug efficacy across nine age groups of PKU patients and evaluate model parameters that are influenced by patient physiology, microbe selection and therapeutic production, and dosing formulations. In particular, the ALT-CAT model describes the mathematical framework to model the behavior of orally delivered engineered bacteria that act as living therapeutics by adapting similar methods that have been developed and widely-used for small molecular drug delivery and absorption.     

Accumulation of sphingomyelin in Niemann-Pick disease type C cells disrupts Rab9-dependent vesicular trafficking of cholesterol

Niemann-Pick disease type C (NPC) is a genetic disorder in which patient cells have endosomal/lysosomal accumulation of cholesterol and sphingolipids. However, the relationship between sphingolipids and cholesterol accumulation in NPC cells has not been established. Here, we investigated the role of sphingomyelin (SM) on the accumulation of cholesterol in NPC cells. Reduction of SM by inhibition of the ceramide transfer protein CERT decreased the cholesterol accumulation in NPC cells. The accumulation of SM in NPC cells inhibited the transport of cholesterol to the endoplasmic reticulum. Overexpression of Rab9 in NPC cells reduced the cholesterol accumulation, which was recovered by treatment with SM. In NPC cells that overexpressed a Rab9 constitutively active mutant, SM treatment did not lead to the cholesterol accumulation. These results indicate that SM negatively regulates the Rab9-dependent vesicular trafficking of cholesterol, and a reduction in SM levels in NPC cells recovers the Rab9-dependent vesicular trafficking defect.     

Urban planning of the endoplasmic reticulum (ER): How diverse mechanisms segregate the many functions of the ER

The endoplasmic reticulum (ER) is the biggest organelle in most cell types, but its characterization as an organelle with a continuous membrane belies the fact that the ER is actually an assembly of several, distinct membrane domains that execute diverse functions. Almost 20 years ago, an essay by Sitia and Meldolesi first listed what was known at the time about domain formation within the ER. In the time that has passed since, additional ER domains have been discovered and characterized. These include the mitochondria-associated membrane (MAM), the ER quality control compartment (ERQC), where ER-associated degradation (ERAD) occurs, and the plasma membrane-associated membrane (PAM). Insight has been gained into the separation of nuclear envelope proteins from the remainder of the ER. Research has also shown that the biogenesis of peroxisomes and lipid droplets occurs on specialized membranes of the ER. Several studies have shown the existence of specific marker proteins found on all these domains and how they are targeted there. Moreover, a first set of cytosolic ER-associated sorting proteins, including phosphofurin acidic cluster sorting protein 2 (PACS-2) and Rab32 have been identified. Intra-ER targeting mechanisms appear to be superimposed onto ER retention mechanisms and rely on transmembrane and cytosolic sequences. The crucial roles of ER domain formation for cell physiology are highlighted with the specific targeting of the tumor metastasis regulator gp78 to ERAD-mediating membranes or of the promyelocytic leukemia protein to the MAM.     

Unusual sterolic mixture,and 24‐isopropylcholesterol,from the sponge Ciocalypta sp. reduce cholesterol uptake and basolateral secretion in Caco‐2 cells

An unusual sterolic mixture (82.3% of 24‐isopropylated sterols) and its major component, 24‐isopropylcholesterol, isolated from a marine sponge, Ciocalypta sp. (Halichondriidae), reduce cholesterol uptake, basolateral secretion and ACAT‐2 mRNA expression and increase the expression of ABCA1 mRNA in Caco‐2 cells. The decreases of cholesterol uptake and secretion induced by 24‐isopropylcholesterol alone were more than that of both the sterolic mixture and β‐sitosterol. These data add a new sterol, 24‐isopropylcholesterol, to sterols that may reduce intestinal cholesterol absorption. J. Cell. Biochem. 106: 659–665, 2009. © 2009 Wiley‐Liss, Inc.     

Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase

Pregnenolone (PREG) can be converted to PREG esters (PE) by the plasma enzyme lecithin: cholesterol acyltransferase (LCAT), and by other enzyme(s) with unknown identity. Acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) convert various sterols to steryl esters; their activities are activated by cholesterol. PREG is a sterol-like molecule, with 3-β-hydroxy moiety at steroid ring A, but with much shorter side chain at steroid ring D. Here we show that without cholesterol, PREG is a poor ACAT substrate; with cholesterol, the V(max) for PREG esterification increases by 100-fold. The binding affinity of ACAT1 for PREG is 30-50-fold stronger than that for cholesterol; however, PREG is only a substrate but not an activator, while cholesterol is both a substrate and an activator. These results indicate that the sterol substrate site in ACAT1 does not involve significant sterol-phospholipid interaction, while the sterol activator site does. Studies utilizing small molecule ACAT inhibitors show that ACAT plays a key role in PREG esterification in various cell types examined. Mice lacking ACAT1 or ACAT2 do not have decreased PREG ester contents in adrenals, nor do they have altered levels of the three major secreted adrenal steroids in serum. Mice lacking LCAT have decreased levels of PREG esters in the adrenals. These results suggest LCAT along with ACAT1/ACAT2 contribute to control pregnenolone ester content in different cell types and tissues.     

17beta-estradiol enhances the flux of cholesterol through the cholesteryl ester cycle in human macrophages

Estrogens have been shown to have many positive effects on the function of arterial wall, and recent evidence suggest that 17-estradiol has a direct action in reducing the accumulation of cholesteryl ester in macrophages. The mechanisms underlying the effects of 17-estradiol on foam cell formation, however are poorly understood. The aim of this study is to investigate the role of 17-estradiol in the regulation of the cholesteryl ester cycle and cholesterol efflux in human macrophages. In addition, the influence of 17-estradiol on apolipoprotein E (apoE) and lipoprotein lipase (LDL) secretion by the cells was also tested. Human Monocyte Derived Macrophages (HMDM), matured in the presence or the absence of 17-estradiol, were loaded with [3H]-cholesteryl ester-labeled-acetyl LDL (low density lipoprotein) and the efflux of radioactivity into the medium was measured. The effect of 17-estradiol on cellular activities of acyl coenzyme A: cholesterol acyl transferase (ACAT), and both neutral and acid cholesteryl ester hydrolase (CEH) and the secretion of apoE and LDL into the medium, were also studied. The results indicate that 17-estradiol induces an increase in the amount of labeled cholesterol released from the cells and, the data obtained from the measurements of ACAT and CEH activities showed that, in estrogen-treated HMDM, the cholesteryl ester cycle favors the hydrolysis of lipoprotein cholesterol by CEH in comparison with its acylation by ACAT. In particular, for the first time a strong enhancement of neutral and acid CEH in human macrophages by 17-estradiol, was demonstrated. ApoE and LDL secretion increased during the maturation of monocytes to macrophages, and was not modified by 17-estradiol. In contrast, loading the cells with cholesterol by incubation in the presence of acetylated or oxidized LDL produced an increase in the levels of apoE secreted by both estrogen-treated and control macrophages. The activity of LPL found in the cell medium, on the other hand, in lipid loaded cells tended to be increased only in estrogen treated macrophages, suggesting that the effects of estrogen on unloaded macrophages are different from those produced on lipid-loaded macrophages. On the whole, the present findings suggest that one of the mechanisms by which 17-estradiol acts to reduce cholesterol accumulation in macrophages is by increasing reverse cholesterol transport through the enhancement of the cholesteryl ester cycle, so that the generation of intracellular unesterified cholesterol for excretion from the cells is favored.     

ACAT1 deletion in murine macrophages associated with cytotoxicity and decreased expression of collagen type 3A1

In contrast to some published studies of murine macrophages, we previously showed that ACAT inhibitors appeared to be anti-atherogenic in primary human macrophages in that they decreased foam cell formation without inducing cytotoxicity. Herein, we examined foam cell formation and cytotoxicity in murine ACAT1 knockout (KO) macrophages in an attempt to resolve the discrepancies. Elicited peritoneal macrophages from normal C57BL6 and ACAT1 KO mice were incubated with DMEM containing acetylated LDL (acLDL, 100 microg protein/ml) for 48h. Cells became cholesterol enriched and there were no differences in the total cholesterol mass. Esterified cholesterol mass was lower in ACAT1 KO foam cells compared to normal macrophages (p<0.04). Cytotoxicity, as measured by the cellular release of [(14)C]adenine from macrophages, was approximately 2-fold greater in ACAT1 KO macrophages as compared to normal macrophages (p<0.0001), and this was independent of cholesterol enrichment. cDNA microarray analysis showed that ACAT1 KO macrophages expressed substantially less collagen type 3A1 (26-fold), which was confirmed by RT-PCR. Total collagen content was also significantly reduced (57%) in lung homogenates isolated from ACAT1 KO mice (p<0.02). Thus, ACAT1 KO macrophages show biochemical changes consistent with increased cytotoxicity and also a novel association with decreased expression of collagen type 3A1.     

Two human ACAT2 mRNA variants produced by alternative splicing and coding for novel isoenzymes

Acyl coenzyme A：cholesterol acyltransferase 2 （ACAT2） plays an important role in cholesterol absorption. Human ACAT2 is highly expressed in small intestine and fetal liver, but its expression is greatly diminished in adult liver. The full-length human ACAT2 mRNA encodes a protein, designated ACAT2a, with 522 amino acids. We have previously reported the organization of the human ACAT2 gene and the differentiation-dependent promoter activity in intestinal Caco-2 cells. In the current work, two human ACAT2 mRNA variants produced by alternative splicing are cloned and predicted to encode two novel ACAT2 isoforms, named ACAT2b and ACAT2c, with 502 and 379 amino acids, respectively. These mRNA variants differ from ACAT2a mRNA by lack of the exon 4 （ACAT2b mRNA） and exons 4-5 plus 8-9-10 （ACAT2c mRNA）. Significantly, comparable amounts of the alternatively spliced ACAT2 mRNA variants were detected by RT- PCR, and Western blot analysis confirmed the presence of their corresponding proteins in human liver and intestine cells. Furthermore, phosphorylation and enzymatic activity analyses demonstrated that the novel isoenzymes ACAT2b and ACAT2c lacked the phosphorylatable site SLLD, and their enzymatic activities reduced to 25%-35% of that of ACAT2a. These evidences indicate that alternative splicing produces two human ACAT2 mRNA variants that encode the novel ACAT2 isoenzymes. Our findings might help to understand the regulation of the ACAT2 gene expression under certain physiological and pathological conditions.