De Novo Cholesterol Research Articles

Impact of De Novo Cholesterol Biosynthesis on the Initiation and Progression of Breast Cancer.

Cholesterol (CHOL) is a multifaceted lipid molecule. It is an essential structural component of cell membranes, where it cooperates in regulating the intracellular trafficking and signaling pathways. Additionally, it serves as a precursor for vital biomolecules, including steroid hormones, isoprenoids, vitamin D, and bile acids. Although CHOL is normally uptaken from the bloodstream, cells can synthesize it de novo in response to an increased requirement due to physiological tissue remodeling or abnormal proliferation, such as in cancer. Cumulating evidence indicated that increased CHOL biosynthesis is a common feature of breast cancer and is associated with the neoplastic transformation of normal mammary epithelial cells. After an overview of the multiple biological activities of CHOL and its derivatives, this review will address the impact of de novo CHOL production on the promotion of breast cancer with a focus on mammary stem cells. The review will also discuss the effect of de novo CHOL production on in situ and invasive carcinoma and its impact on the response to adjuvant treatment. Finally, the review will discuss the present and future therapeutic strategies to normalize CHOL biosynthesis.

LH Induces De Novo Cholesterol Biosynthesis via SREBP Activation in Granulosa Cells During Ovulation in Female Mice.

In the liver, the sterol response element binding protein (SREBP) and the SREBP cleavage-activated protein (SCAP) complex upregulate cholesterol biosynthesis by gene induction of de novo cholesterol synthetic enzymes (Hmgcr, Cyp51, and Dhcr7). Insulin induced gene 1 (INSIG1) negatively regulates cholesterol biosynthesis by the inhibition of de novo cholesterol biosynthetic gene expression. In the ovary, cholesterol is de novo synthesized; however, the roles of SREBP and its regulators (SCAP and INSIG1) are not well understood. In this study, when immature mice were treated with gonadotropins (eCG followed by hCG), eCG induced and hCG maintained the expression of SREBP-1a, -2, and SCAP granulosa cells, whereas INSIG1 expression was dramatically downregulated after hCG injection. Downregulation of INSIG1 led to generate the SREBPs active form and translocate the SREBPs active form to nuclei. Inhibition of generation of the SREBPs active form by fatostatin or Scap siRNA in both in vivo and in vitro significantly decreased the expressions of de novo cholesterol biosynthetic enzymes, cholesterol accumulation, and progesterone (P4) production compared with the control group. Fatostatin treatment inhibited the ovulation and increased the formation of abnormal corpus luteum which trapped the matured oocyte in the corpus luteum; however, the phenomenon was abolished by P4 administration. The results showed that decreasing INSIG1 level after hCG stimulation activated SREBP-induced de novo cholesterol biosynthesis in granulosa cells of preovulatory follicles, which is essential for P4 production and the rupture of matured oocyte during ovulation process.

Disruption of cholesterol metabolism via sigma-2 ligands induces cell death in pancreatic cancer

Presenter: Rony Takchi MD | Washington University, St. Louis Background: The malignant transformation of pancreatic ductal adenocarcinoma (PDAC) is accompanied by alteration of the metabolism to adapt to the new hypoxic and nutrient-deprived micro-environment. Recent studies have shown that PDAC is highly dependent on cholesterol import. Consequently, tumors with a high LDL-receptor (LDL-R) expression phenotype are more aggressive, which is linked with worse patient survival. Like other proliferating tumors, PDAC is characterized by overexpression of the sigma-2 receptor, which is involved in enhanced LDL-R activity and the regulation of cholesterol export from the lysosomes in an NPC1-dependent manner. We thus hypothesized that sigma-2 ligands may facilitate pancreatic cancer cell death via cholesterol trapping in the lysosomes through disruption of the NPC1-mediated export mechanism. Methods: The sigma-2 ligands tested are the azabicyclononane analogs SW43, SW96, SV95, and the benzamide analog, ISO-1. To determine the kinetics and the cellular localization properties of a representative member of the sigma-2 ligand family, SW43 was chemically linked to the fluorophore 4-Chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl; SW120). To study the impact on cellular cholesterol trafficking upon sigma-2 ligand treatment, cells were stained with Filipin and analyzed via confocal microscopy. Protein abundance of key players involved in intracellular cholesterol processing/trafficking was determined using state-of-the-art WES technology employing the following antibodies: Sterol-regulatory binding protein 2 (SREBP2), LDL-R, Niemann-Pick C1 protein (NPC1). Cell viability was determined using IncuCyte live-cell analysis system. In order to study the predicted synergism between cholesterol sequestration and blocking of de novo cholesterol synthesis, PDAC cells were treated with sigma-2 ligands in combination with simvastatin, which inhibits the rate-limiting enzyme in cholesterol synthesis 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAr). Drug combination effects (synergy) were determined using CompuSyn software. Results: Utilizing three different human pancreatic cancer cell lines (AsPC1, HPAC, and MiaPaca-2), fluorescently labeled sigma-2 ligand SW120 was shown to be rapidly taken up into the lysosomal compartment within minutes. This rapid uptake mechanism was associated with a moderate cytotoxicity profile of the sigma-2 ligands SW43 and SW96. Of note, the sigma-2 ligand-mediated cytotoxicity correlated with a rapid cholesterol sequestration phenotype in the lysosomes. In contrast, treatment with two other, high-affinity sigma-2 ligands SV95 and ISO1, was not associated with cytotoxicity and did not lead to cholesterol sequestration. Since trapping of cholesterol in the lysosomes renders it unusable for the cell, analogous to the lysosomal storage disease Niemann-Pick type C, actuation of the sterol sensing machinery was predicted. And indeed, shortly (time) after exposure to SW43 or SW96, the cholesterol sensor released SREBP-2 which in turn was activated and induced production of its downstream targets, LDL-R and NPC1. Moreover, the combination of cholesterol sequestration (SW43) and blocking of de novo cholesterol production (simvastatin), demonstrated a synergistic drug activity profile. Conclusion: We identified a cholesterol trapping phenotype as an early event of sigma-2 ligand-mediated cytotoxicity. Targeting both LDL-R-mediated lysosomal cholesterol trapping with sigma-2 ligands in combination with blockage of de-novo cholesterol synthesis lead to synergistic cancer cell death and thus represents an intriguing novel treatment concept for PDAC.

Sa2009 – Combinatorial Blockade of De Novo Cholesterol Biosynthesis and Pcsk9 As a Synergistic Therapy Fro KrasMutant Colorectal Cancer

Sa2009 – Combinatorial Blockade of De Novo Cholesterol Biosynthesis and Pcsk9 As a Synergistic Therapy Fro KrasMutant Colorectal Cancer

Transcriptional regulation of lipid metabolism when salmon fry switches from endogenous to exogenous feeding

The onset of first feeding is a critical period for Atlantic salmon (Salmo salar). The fish goes through a dramatic transition, from using the yolk sac as an energy and nutrient resource to rely on exogenous food. However, the digestive system of salmon is not fully developed at start feeding. This is especially true for digestion and absorption of dietary lipids. To optimize lipid composition of the initial feed for better growth and development of salmon, it is important to have a systemic understanding of lipid metabolism. The present study combines transcriptomics and lipid composition data to obtain an overview of how lipid metabolism pathways change when salmon switch from endogenous to exogenous feed. Transition to exogenous feeding had higher influence on gene expression and fatty acids composition in pyloric caeca relative to liver, suggesting metabolism is more responsive in pyloric caeca when diet switches. The pathways of phospholipid and lipoprotein synthesis were both up-regulated in pyloric caeca, while phospholipid content unchanged. Because phospholipids are a major component of intestinal lipoproteins, it suggests that a higher level of dietary phospholipids is required for optimal lipid transport at first feeding stages. De-novo cholesterol synthesis pathway was up-regulated in pyloric caeca after exogenous feeding, while a down-regulation of bile acid synthesis pathway was found in liver. This suggests a higher requirement of cholesterol in salmon fry after switching to exogenous feeding. The present study has provided new insights on the systemic changes of lipid synthesis and transport pathways in salmon fry when switching from endogenous to exogenous feeding.

Altered membrane rigidity via enhanced endogenous cholesterol synthesis drives cancer cell resistance to destruxins.

Destruxins, secondary metabolites of entomopathogenic fungi, exert a wide variety of interesting characteristics ranging from antiviral to anticancer effects. Although their mode of action was evaluated previously, the molecular mechanisms of resistance development are unknown. Hence, we have established destruxin-resistant sublines of HCT116 colon carcinoma cells by selection with the most prevalent derivatives, destruxin (dtx)A, dtxB and dtxE. Various cell biological and molecular techniques were applied to elucidate the regulatory mechanisms underlying these acquired and highly stable destruxin resistance phenotypes. Interestingly, well-known chemoresistance-mediating ABC efflux transporters were not the major players. Instead, in dtxA- and dtxB-resistant cells a hyper-activated mevalonate pathway was uncovered resulting in increased de-novo cholesterol synthesis rates and elevated levels of lanosterol, cholesterol as well as several oxysterol metabolites. Accordingly, inhibition of the mevalonate pathway at two different steps, using either statins or zoledronic acid, significantly reduced acquired but also intrinsic destruxin resistance. Vice versa, cholesterol supplementation protected destruxin-sensitive cells against their cytotoxic activity. Additionally, an increased cell membrane adhesiveness of dtxA-resistant as compared to parental cells was detected by atomic force microscopy. This was paralleled by a dramatically reduced ionophoric capacity of dtxA in resistant cells when cultured in absence but not in presence of statins. Summarizing, our results suggest a reduced ionophoric activity of destruxins due to cholesterol-mediated plasma membrane re-organization as molecular mechanism underlying acquired destruxin resistance in human colon cancer cells. Whether this mechanism might be valid also in other cell types and organisms exposed to destruxins e.g. as bio-insecticides needs to be evaluated.

Genomic profiling of bovine corpus luteum maturation.

To unveil novel global changes associated with corpus luteum (CL) maturation, we analyzed transcriptome data for the bovine CL on days 4 and 11, representing the developing vs. mature gland. Our analyses revealed 681 differentially expressed genes (363 and 318 on day 4 and 11, respectively), with ≥2 fold change and FDR of <5%. Different gene ontology (GO) categories were represented prominently in transcriptome data at these stages (e.g. days 4: cell cycle, chromosome, DNA metabolic process and replication and on day 11: immune response; lipid metabolic process and complement activation). Based on bioinformatic analyses, select genes expression in day 4 and 11 CL was validated with quantitative real-time PCR. Cell specific expression was also determined in enriched luteal endothelial and steroidogenic cells. Genes related to the angiogenic process such as NOS3, which maintains dilated vessels and MMP9, matrix degrading enzyme, were higher on day 4. Importantly, our data suggests day 11 CL acquire mechanisms to prevent blood vessel sprouting and promote their maturation by expressing NOTCH4 and JAG1, greatly enriched in luteal endothelial cells. Another endothelial specific gene, CD300LG, was identified here in the CL for the first time. CD300LG is an adhesion molecule enabling lymphocyte migration, its higher levels at mid cycle are expected to support the transmigration of immune cells into the CL at this stage. Together with steroidogenic genes, most of the genes regulating de-novo cholesterol biosynthetic pathway (e.g HMGCS, HMGCR) and cholesterol uptake from plasma (LDLR, APOD and APOE) were upregulated in the mature CL. These findings provide new insight of the processes involved in CL maturation including blood vessel growth and stabilization, leucocyte transmigration as well as progesterone synthesis as the CL matures.

Hydroxysteroid (17β) Dehydrogenase 7 Activity Is Essential for Fetal de Novo Cholesterol Synthesis and for Neuroectodermal Survival and Cardiovascular Differentiation in Early Mouse Embryos

Hydroxysteroid (17beta) dehydrogenase 7 (HSD17B7) has been shown to catalyze the conversion of both estrone to estradiol (17-ketosteroid reductase activity) and zymosterone to zymosterol (3-ketosteroid reductase activity involved in cholesterol biosynthesis) in vitro. To define the metabolic role of the enzyme in vivo, we generated knockout mice deficient in the enzyme activity (HSD17B7KO). The data showed that the lack of HSD17B7 results in a blockage in the de novo cholesterol biosynthesis in mouse embryos in vivo, and HSD17BKO embryos die at embryonic day (E) 10.5. Analysis of neural structures revealed a defect in the development of hemispheres of the front brain with an increased apoptosis in the neuronal tissues. Morphological defects in the cardiovascular system were also observed from E9.5 onward. Mesodermal, endodermal, and hematopoietic cells were all detected by the histological analysis of the visceral yolk sac, whereas no organized vessels were observed in the knockout yolk sac. Immunohistological staining for platelet endothelial cell adhesion molecule-1 indicated that the complexity of the vasculature also was reduced in the HSD17B7KO embryos, particularly in the head capillary plexus and branchial arches. At E8.5-9.5, the heart development and the looping of the heart appeared to be normal in the HSD17B7KO embryos. However, at E10.5 the heart was dilated, and the thickness of the cardiac muscle and pericardium in the HSD17B7KO embryos was markedly reduced, and immunohistochemical staining for GATA-4 revealed that HSD17B7KO embryos had a reduced number of myocardial cells. The septum of the atrium was also defected in the knockout mice.

Activation of Liver X Receptors Prevents Statin-induced Death of 3T3-L1 Preadipocytes

The biological functions of liver X receptors (LXRs) alpha and beta have primarily been linked to pathways involved in fatty acid and cholesterol homeostasis. Here we report a novel role of LXR activation in protecting cells from statin-induced death. When 3T3-L1 preadipocytes were induced to differentiate by standard isobutylmethylxanthine/dexamethasone/insulin treatment in the presence of statins, they failed to differentiate and underwent massive apoptosis. The simultaneous addition of selective LXR agonists prevented the statin-induced apoptosis. By using mouse embryo fibroblasts from wild-type (LXRalpha+/+/LXRbeta+/+), LXRalpha knock-out mice (LXRalpha(-/-)/LXRbeta+/+), LXRbeta knock-out mice (LXRalpha+/-/LXRbeta(-/-)), and LXR double knock-out mice (LXRalpha(-/-)/LXRbeta(-/-)) as well as 3T3-L1 cells transduced with retroviruses expressing either wild-type LXRalpha or a dominant negative version of LXRalpha, we demonstrate that the response to LXR agonists is LXR-dependent. Interestingly, LXR-mediated rescue of statin-induced apoptosis was not related to up-regulation of genes previously shown to be involved in the antiapoptotic action of LXR. Furthermore, forced expression of Bcl-2 did not prevent statin-induced apoptosis; nor did LXR action depend on protein kinase B, whose activation by insulin was impaired in statin-treated cells. Rather, LXR-dependent rescue of statin-induced apoptosis in 3T3-L1 preadipocytes required NF-kappaB activity, since expression of a dominant negative version of IkappaBalpha prevented LXR agonist-dependent rescue of statin-induced apoptosis. Thus, the results presented in this paper provide novel insight into the action of statins on and LXR-dependent inhibition of apoptosis.

Topical Mevalonic Acid Stimulates De Novo Cholesterol Synthesis and Epidermal Permeability Barrier Homeostasis in Aged Mice

Extracellular lipids of the stratum corneum, which are composed of cholesterol, fatty acid, and ceramides, are essential for the epidermal permeability barrier function. With damage to the barrier, a decreased capacity for epidermal lipid biosynthesis in aged epidermis results in an impaired repair response. Mevalonic acid is an intermediate after the rate-limiting step in cholesterol biosynthesis, which is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase. In the present study, we investigated the effect of topical mevalonic acid on the murine epidermal permeability barrier function, comparing it with that of cholesterol. Topical treatment with acetone caused linear increases in transepidermal water loss, in proportion to the number of treatments more rapidly in aged mice than in young mice. Administration of mevalonic acid on aged murine epidermis enhanced its resistance against damage and the recovery rate of barrier function from acute barrier disruption. In contrast, although cholesterol also had the same effect, it required a much higher amount than mevalonic acid. In young mice, neither mevalonic acid nor cholesterol had any effect on resistance against acetone damage nor the recovery rate from acetone damage. In the skin of mice topically administered with mevalonic acid, stimulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were both observed, whereas none was seen with stimulation by equimolar cholesterol. These data indicate that a topical application of mevalonic acid enhances barrier recovery in aged mice, which is accompanied by not only acceleration of cholesterol synthesis from mevalonic acid but also stimulation of the whole cholesterol biosynthesis.

Altered Cholesterol Synthesis as a Mechanism Involved in Methyl Isobutyl Ketone-Potentiated Experimental Cholestasis

Mechanisms by which ketones potentiate manganese–bilirubin (Mn–BR)-induced cholestasis are unknown. The purpose of the present study was to investigate the effect of methyl isobutyl ketone (MiBK), a widely used ketonic solvent, at the level of the bile canalicular membrane (BCM) and to verify if altered membrane lipid dynamics could be involved in MiBK-potentiated Mn–BR cholestasis. Male Sprague–Dawley rats were exposed 4 hr/day for 3 days to MiBK vapors (200 or 600 ppm). Eighteen hours after the last exposure, manganese (Mn, 4.5 mg/kg) was given iv followed 15 min later by bilirubin (BR, 25 mg/kg). Rats were killed 30 min after BR; liver cell plasma membranes (bile canalicular and sinusoidal), microsomes, mitochondria, and cytosol were isolated by differential centrifugation. Lipids were extracted and cholesterol was measured in each fraction. After Mn–BR and MiBK exposure (600 ppm), results indicated a marked increase in BCM cholesterol content compared to rats exposed to air only. This increase was greater than that due to Mn–BR or MiBK given alone. Also, results indicated that cholesterol increased in a dose-related fashion in BCM after MiBK exposure, whereas PM cholesterol remained unaltered. To identify the source of the increased BCM cholesterol and to permit distinction between de novocholesterol synthesis and subcellular shifts, the hepatic lipid pool was labeled in vivowith [ 3H]-cholesterol and [2- 14C]-mevalonic acid, a cholesterol synthesis precursor. Results showed that after 600 ppm MiBK exposure, 14C-labeled cholesterol was greater than 3H-labeled cholesterol, indicating that the contribution of de novocholesterol synthesis to the total cholesterol content of the various isolated hepatocellular fractions was more important than the contribution of intracellular pools. Therefore, increased BCM cholesterol content and enhanced accumulation of newly synthesized cholesterol appear to be involved in MiBK potentiation of Mn–BR-induced cholestasis.

Regulation of steroidogenesis and cholesterol synthesis by prostaglandin F-2 alpha and lipoproteins in bovine luteal cells.

Bovine luteal cells can utilize low density lipoprotein (LDL) or high density lipoprotein (HDL) as a source of cholesterol for steroidogenesis, and administration of PGF-2 alpha in vitro suppresses lipoprotein utilization. The objective of this study was to examine the mechanism by which PGF-2 alpha exerts this effect. Cultured bovine luteal cells received 0.25 microCi[14C]acetate/ml, to assess rates of de-novo sterol and steroid synthesis, with or without lipoproteins. Both LDL and HDL enhanced progesterone production (P less than 0.01), but caused a significant reduction in the amount of radioactivity in the cholesterol fraction. PGF-2 alpha treatment inhibited the increase in lipoprotein-induced progesterone synthesis (P less than 0.01), but did not prevent the reduction in de-novo cholesterol synthesis brought about by LDL or HDL. PGF-2 alpha alone reduced cholesterol synthesis (P less than 0.01), but it was not as effective as either LDL or HDL. Both lipoproteins and PGF-2 alpha also decreased the amount of radioactivity in the progesterone fraction (P less than 0.01), and the effect of PGF-2 alpha was similar to that of the lipoproteins. It is concluded that lipoproteins can enhance progesterone production and also suppress de-novo cholesterol synthesis in bovine luteal cells, but only the former effect of lipoproteins is inhibited by PGF-2 alpha. Therefore, it is suggested that PGF-2 alpha allows entry of lipoprotein cholesterol into the cell, but prevents utilization for steroidogenesis. In addition, PGF-2 alpha alone can suppress cholesterol synthesis, as well as decrease conversion of cholesterol to progesterone.

Role of lipoproteins and de-novo cholesterol synthesis in progesterone production by cultured bovine luteal cells

Summary. Bovine luteal cells cultured in the presence of lipoprotein-deficient serum (LPDS) produced less progesterone than did cells cultured in the presence of complete serum and, while dibutyryl cyclic AMP (dbcAMP) caused a marked stimulation of progesterone production in complete serum, it had only a small effect in LPDS. Lowdensity lipoprotein (LDL) and high-density lipoprotein (HDL) increased basal and dbcAMP-stimulated progesterone production. Cells were 7 times more sensitive to LDL than HDL although the maximum response to both lipoproteins did not differ significantly. Inhibition of de-novo cholesterol synthesis by compactin had no effect on dbcAMP-stimulated progesterone production by cells cultured with complete serum or LPDS plus lipoproteins. Basal progesterone production in complete serum was increased by compactin and this was associated with a marked inhibition of cell proliferation, both effects being reversed by mevalonic acid. Compactin caused a 50% reduction in dbcAMP-stimulated progesterone secretion by cells cultured in LPDS. These data show that bovine luteal cells are dependent upon lipoproteins to provide substrate for progesterone synthesis and that, while LDL is the preferred lipoprotein, both LDL and HDL may be of importance in vivo.

Differences in de novo cholesterol synthesis between the intact male and female rat.

Three different isotopes were used to quantify de novo sterologenesis in intact male and female rats. All three substrates (i.e. [14C]acetate, [14C]octanoate, and [3H]water) were incorporated into nonsaponifiable lipids and cholesterol at significantly greater rates in males than in females. Even with cholesterol feeding, male animals synthesized significantly more cholesterol and nonsaponifiable lipids than females. The primary site of this sex difference in sterologenesis is extrahepatic, extraintestinal tissues (e.g. carcass). In the carcass this sex difference is chiefly due to an enhancement of sterol synthesis in the skin of male rats. Cholesterol synthesis is 73% greater and nonsaponifiable lipid synthesis is 85% greater in the skin of males than in females. Moreover, de novo sterologenesis in skin is hormonally dependent. In castrated females, testosterone treatment results in a 2-fold stimulation of skin sterol synthesis compared to that in animals administered estradiol or oil vehicle alone. In castrated males, estradiol treatment caused a 30% reduction in skin cholesterol and nonsaponifiable lipid synthesis compared to that in animals administered testosterone. The effects of sex steroid hormones on skin are, therefore, probably responsible for mediating the observed sex difference in de novo sterol synthesis. Additionally, this study demonstrates that the administration of estradiol and testosterone in physiological doses to castrated animals has no effect on cholesterol or nonsaponifiable lipid synthesis in liver, intestine, or other nondermal tissues.

Follicular fluid stimulation of progesterone secretion: time course, dose-response and effect of inhibiting de novo cholesterol synthesis.

This study extends our previous observations of follicular fluid stimulation of progesterone secretion by granulosa cells from immature porcine antral follicles in vitro. Granulosa cells were incubated with either charcoal-treated fluid from large porcine follicles or serum in TC medium 199. Within minutes of the addition of follicular fluid to the cells, enhanced progesterone secretion was observed. A second, more substantial enhancement of progesterone secretion was observed after an additional incubation of 24 to 48 h. As we were unable to detect significant amounts of progesterone within either freshly collected or incubated cells, follicular fluid appears to stimulate both the synthesis and secretion of progesterone rather than just the secretion of preformed progesterone. Inhibition of de novo cholesterol synthesis with an inhibitor of the conversion of 7-dehydrocholesterol to cholesterol, reduced progesterone secretion by cells incubated with serum to a greater extent than by cells incubated with follicular fluid. These studies suggest that follicular fluid may stimulate both de novo cholesterol synthesis and progesterone synthesis from preformed cholesterol. A dose-response test for follicular fluid stimulation of progesterone secretion indicated that 30% follicular fluid in TC medium 199 was as stimulatory as 50% follicular fluid. Ten percent follicular fluid was not stimulatory by itself, but in combination with 1 microgram of FSH/ml (ovine follicle-stimulating hormone), stimulated progesterone secretion to a greater extent than 30% or 50% follicular fluid alone.