Surprising unreactivity of cholesterol-5,6-epoxides towards nucleophiles

We recently established that drugs used for the treatment and the prophylaxis of breast cancers, such as tamoxifen, were potent inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), which led to the accumulation of 5,6α-epoxy-cholesterol (5,6α-EC) and 5,6β-epoxy-cholesterol (5,6β-EC). This could be considered a paradox because epoxides are known as alkylating agents with putative carcinogenic properties. We report here that, as opposed to the carcinogen styrene-oxide, neither of the ECs reacted spontaneously with nucleophiles. Under catalytic conditions, 5,6β-EC remains unreactive whereas 5,6α-EC gives cholestan-3β,5α-diol-6β-substituted compounds. These data showed that 5,6-ECs are stable epoxides and unreactive toward nucleophiles in the absence of a catalyst, which contrasts with the well-known reactivity of aromatic and aliphatic epoxides. These data rule out 5,6-EC acting as spontaneous alkylating agents. In addition, these data support the existence of a stereoselective metabolism of 5,6α-EC.     

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.     

One step synthesis of 6-oxo-cholestan-3β,5α-diol

Cholesterol metabolism has been recently linked to cancer, highlighting the importance of the characterization of new metabolic pathways in the sterol series. One of these pathways is centered on cholesterol-5,6-epoxides (5,6-ECs). 5,6-ECs can either generate dendrogenin A, a tumor suppressor present in healthy mammalian tissues, or the carcinogenic cholestane-3β,5α,6β-triol (CT) and its putative metabolite 6-oxo-cholestan-3β,5α-diol (OCDO) in tumor cells. We are currently investigating the identification of the enzyme involved in OCDO biosynthesis, which would be highly facilitated by the use of commercially unavailable [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol. In the present study we report the one-step synthesis of [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol by oxidation of [¹⁴C]-cholesterol with iodide metaperiodate (HIO₄).     

A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells

Since over 60% of breast cancers are estrogen receptor positive (ER+), many therapies have targeted the ER. The ER is activated by both estrogen binding and phosphorylation. While anti-estrogen therapies, such as tamoxifen (Tam) have been successful they do not target the growth factor promoting phosphorylation of the ER. Other proliferation pathways such as the phosphatidylinositol-3 kinase, (PI3K) and the mitogen-activated protein kinase (MAPK) pathways are activated in breast cancer cells and are associated with poor prognosis. Thus targeting multiple cellular proliferation and survival pathways at the onset of treatment is critical for the development of more effective therapies. The grapefruit flavanone naringenin (Nar) is an inhibitor of both the PI3K and MAPK pathways. Previous studies examining either Nar or Tam used charcoal-stripped serum which removed estrogen as well as other factors. We wanted to use serum containing medium in order to retain all the potential inducers of cell proliferation so as not to exclude any targets of Nar. Here we show that a Nar–Tam combination is more effective than either Tam alone or Nar alone in MCF-7 breast cancer cells. We demonstrate that a Nar–Tam combination impaired cellular proliferation and viability to a greater extent than either component alone in MCF-7 cells. Furthermore, the use of a Nar–Tam combination requires lower concentrations of both compounds to achieve the same effects on proliferation and viability. Nar may function by inhibiting both PI3K and MAPK pathways as well as localizing ERα to the cytoplasm in MCF-7 cells. Our results demonstrate that a Nar–Tam combination induces apoptosis and impairs proliferation signaling to a greater extent than either compound alone. These studies provide critical information for understanding the molecular mechanisms involved in cell proliferation and apoptosis in breast cancer cells.     

Direct,Differential Effects of Tamoxifen, 4-Hydroxytamoxifen,and Raloxifene on Cardiac Myocyte Contractility and Calcium Handling

Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT), and related drug raloxifene (Ral) on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca²⁺ transient amplitude. Effects were primarily observed at 5 and 10 μM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca²⁺ transient amplitudes. At 10 μM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca²⁺ handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy.     

Increased expression of peripheral benzodiazepine receptor (PBR) in dimethylbenz[a]anthracene-induced mammary tumors in rats

Expression of peripheral benzodiazepine receptors (PBR) has been found in every tissue examined; however, it is most abundant in steroid-producing tissues. Although the primary function of PBR is the regulation of steroidogenesis, its existence in nonsteroidogenic tissues as well as in other cellular compartments including the nucleus suggests that there may be other roles for PBR. Our laboratory reported earlier a significant increase of PBR density in the nucleus of DMBA-induced malignant submandibular glands of rats, suggesting a role of PBR in nuclear events of peripheral tissues. Since then numerous studies have demonstrated the abundance of PBR in tumors. Numerous studies implicate a role for cholesterol in the mechanisms underlying cell proliferation and cancer progression. Based on studies with a battery of human breast cancer cell lines and several human tissue biopsies, Hardwick et al. suggested that PBR expression, nuclear localization, and PBR-mediated cholesterol transport into the nucleus are involved in human breast cancer cell proliferation and aggressive phenotype expression. The purpose of the present study is to confirm this hypothesis by developing an animal breast cancer model and correlating the above events with the breast cancer. Weanling rats were maintained on a diet containing animal protein (casein) for 30 days and then a single dose of DMBA in sesame oil (80 mg/kg) was administered by gavage to the animals. Control animals received the vehicle only. After 122 days of DMBA administration, the animals were sacrificed. All tumors were detected by palpation. B_max of PBRs was 52.6% and 128.4% higher in the non-aggressive and aggressive cancer tissues, respectively, than that in normal tissues. Cholesterol uptake into isolated nuclei was found to be higher in both non-aggressive and aggressive tumor breast tissue than that in control tissue. There was also corresponding increase in B_max of PBRs in the nucleus of cancer tissues. Furthermore, the nuclear nucleoside triphosphatase (NTPase) activity was found to be higher in aggressive tumor tissues than that in non-aggressive tumor tissues. In conclusion, these data suggest that PBR ligand binding, and PBR-mediated cholesterol transport into the nucleus may be involved in the development of mammary gland adenocarcinoma, thus participating in the advancement of the disease.     

Metabolism of cholesteryl palmitate by rat brain in vitro; formation of cholesterol epoxides and cholestane-3beta,5alpha,6beta-triol

Incubation of [4-(14)C]cholesteryl palmitate with the 12,000 g supernatant fraction of adult rat brain fortified with an NADPH-generating system and beta-mercaptoethylamine resulted in formation (2-5%) of more polar metabolites characterized as a mixture of cholesterol-5,6-epoxides. Under extended incubation conditions, cholestane-3beta-5alpha-6beta-triol was isolated as the major end product of the incubations. Free [4-(14)C]cholesterol incubated under similar conditions was not oxidized, whereas oxidation of [4-(14)C]cholesteryl palmitate appeared to be dependent upon hydrolysis of the ester by the rat brain microsomal subcellular fraction. Elimination of the NADPH-generating system or the addition of EDTA to the incubation mixture inhibited epoxide formation, suggesting that the products are derived from an NADPH-dependent enzymatic lipoperoxidation mechanism. The in vitro conversion of [4-(14)C]cholesterol-5alpha,6alpha-epoxide to cholestane-3beta,5alpha,6beta-triol was also demonstrated in rat brain subcellular fractions in the absence of added cofactors.     

Cytoplasmic PELP1 and ERRgamma Protect Human Mammary Epithelial Cells from Tam-Induced Cell Death

Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman''s risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.     

Expression of Peripheral Benzodiazepine Receptor (PBR) in Human Tumors: Relationship to Breast,Colorectal, and Prostate Tumor Progression

Abstract

High levels of peripheral‐type benzodiazepine receptor (PBR), the alternative‐binding site for diazepam, are part of the aggressive human breast cancer cell phenotype in vitro. We examined PBR levels and distribution in normal tissue and tumors from multiple cancer types by immunohistochemistry. Among normal breast tissues, fibroadenomas, primary and metastatic adenocarcinomas, there is a progressive increase in PBR levels parallel to the invasive and metastatic ability of the tumor (p < 0.0001). In colorectal and prostate carcinomas, PBR levels were also higher in tumor than in the corresponding non‐tumoral tissues and benign lesions (p < 0.0001). In contrast, PBR was highly concentrated in normal adrenal cortical cells and hepatocytes, whereas in adrenocortical tumors and hepatomas PBR levels were decreased. Moreover, malignant skin tumors showed decreased PBR expression compared with normal skin. These results indicate that elevated PBR expression is not a common feature of aggressive tumors, but rather may be limited to certain cancers, such as those of breast, colon‐rectum and prostate tissues, where elevated PBR expression is associated with tumor progression. Thus, we propose that PBR overexpression could serve as a novel prognostic indicator of an aggressive phenotype in breast, colorectal and prostate cancers.     

Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells

Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone-responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam-mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6-associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome-mediated degradation. Furthermore, we show that Tam- and siE6AP-mediated inhibition of E6AP leads to enhanced G0-G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome-c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam-targeted E6AP inhibition is in fact required for Tam-mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer.     

CB1 and CB2 receptors are novel molecular targets for Tamoxifen and 4OH-Tamoxifen

Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9–3 μM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs.     

Cholesterol-induced growth stimulation, macromolecule synthesis, and increased phosphoinositide metabolism of ascites tumour cells in culture

Ascites tumour cells have previously been shown by us to require exogenous cholesterol for growth. To investigate further this phenomenon, we have used, in addition to free cholesterol, cholesterol complexed to digitonine, to elaborate the specificity of this growth-controlling process using a chemically defined medium. Our data show that only free cholesterol stimulates cell growth and macromolecule synthesis in a dose-dependent manner, suggesting that the proper embedding of the sterol into the membrane is a prerequisite for its function. Furthermore, studies have been performed on the influence of cholesterol on the phosphoinositide metabolism of our cells, as phosphoinositides furnish important second messenger molecules in the cascade of signal transduction. We could show that cholesterol stimulates a transient release of inositol trisphosphate and other inositol phosphates by inducing the activation of phospholipase C (PLC). PLC activation by a factor of about 3 with phosphatidylinositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate as substrates could be measured directly by using a partially purified membrane preparation. This enzyme activity was found to be strongly dependent on free Ca2+ ions with optimal concentrations of 100 nM for cholesterol- and 50 nM for cholesterol-digitonide-treated cells. Ca2+ concentration for half-maximum activation, however, was identical under both conditions. Phospholipase C activity could be synergistically increased about 2-fold with 25 microg GTP gammaS in cholesterol-digitonide-treated cells as well, suggesting that the coupling between phospholipase C and the G-protein was not disturbed by the complex. These data demonstrate a functional role of cholesterol on cell growth, macromolecule synthesis, and phosphoinositide metabolism mediating the release of important second messenger molecules.     

Role of mitochondria in tamoxifen-induced rapid death of MCF-7 breast cancer cells

Tamoxifen (Tam) is widely used in chemotherapy of estrogen receptor-positive breast cancer. It inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor-dependent modulation of gene expression, but recent reports have shown that Tam (especially at pharmacological concentrations) has also rapid nongenomic effects. Here we studied the mechanisms by which Tam exerts rapid effects on breast cancer cell viability. In serum-free medium 5–7 μM Tam induced death of MCF-7 and MDA-MB-231 cells in a time-dependent manner in less than 60 min. This was associated with release of mitochondrial cytochrome c, a decrease of mitochondrial membrane potential and an increase in production of reactive oxygen species (ROS). This suggests that disruption of mitochondrial function has a primary role in the acute death response of the cells. Accordingly, bongkrekic acid, an inhibitor of mitochondrial permeability transition, was able to protect MCF-7 cells against Tam. Rapid cell death induction by Tam was not associated with immediate activation of caspase-9 or cleavage of poly (ADP-ribose) polymerase. It was not blocked by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone either. Diphenylene ionodium (DPI), an inhibitor of NADPH oxidase, was able to prevent Tam-induced cell death but not cytochrome c release, which suggests that ROS act distal to cytochrome c. The pure antiestrogen ICI 182780 (1 μM) could partly oppose the effect of Tam in estrogen receptor positive MCF-7 cells, but not in estrogen receptor negative MDA-MB-231 cells. Pre-culturing MCF-7 cells in the absence of 17β-estradiol (E₂) or in the presence of a low Tam concentration (1 μM) made the cells even more susceptible to rapid death induction by 5 or 7 μM Tam. This effect was associated with decreased levels of the anti-apoptotic proteins Bcl-X_L and Bcl-2. In conclusion, our results demonstrate induction of a rapid mitochondrial cell death program in breast cancer cells at pharmacological concentrations of Tam, which are achievable in tumor tissue of Tam-treated breast cancer patients. These mechanisms may contribute to the ability of Tam therapy to induce death of breast cancer cells.     

The biochemical alterations following administration of Kalpaamruthaa and Semecarpus anacardium in mammary carcinoma

BACKGROUND: Breast cancer is one of the most common cancers in women of developed and developing countries. Lipids, lipoproteins and lipid-metabolizing enzymes have been associated with the risk of breast cancer. Kalpaamruthaa (KA) is a modified Siddha preparation, which contains Semecarpus anacardium Linn. (SA), Emblica officinalis (EO) and honey. OBJECTIVE: The present study was embarked to study the variations in lipids, lipid-metabolizing enzymes and lipoproteins in cancerous animals and the effect of KA on the lipid metabolism. MATERIALS AND METHODS: Breast cancer was induced in rats by administrating 7,12-dimethylbenz(a)anthracene orally (25 mg/kg body weight). After 90 days of induction, KA (300 mg/kg body weight) and SA (200 mg/kg body weight) were administered for 14 days, by gastric intubations. The levels of lipids and lipid-metabolizing enzymes were analysed in control and experimental animals. RESULTS AND CONCLUSION: The increased levels of total cholesterol, free cholesterol, phospholipids, triglycerides and free fatty acids and decreased levels of ester cholesterol in plasma, liver and kidney found in cancer suffering animals were reverted back to near normal levels on treatment with KA and SA. In mammary carcinoma bearing animals, the activities of total lipase, cholesterol ester synthase, and cholesterol ester hydrolase were significantly (p < 0.05) increased whereas lipoprotein lipase and lecithin cholesterol-acyl transferase were decreased. The levels of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were increased and the level of high-density lipoprotein (HDL) was decreased. These alterations were recouped back upon treatment with KA as well as SA when compared to cancer animals. The effects of KA were found to be more effective than SA. No significant alterations were observed in herbal preparation control animals when compared to control animals.     

Pathogens, toxins, and lipid rafts

Summary The plasma membrane is not a uniform two-dimensional space but includes various types of specialized regions containing specific lipids and proteins. These include clathrin-coated pits and caveolae. The existence of other cholesterol- and glycosphingolipid-rich microdomains has also been proposed. The aim of this review is to illustrate that these latter domains, also called lipid rafts, may be the preferential interaction sites between a variety of toxins, bacteria, and viruses and the target cell. These pathogens and toxins have hijacked components that are preferentially found in rafts, such as glycosylphosphatidylinositol-anchored proteins, sphingomyelin, and cholesterol. These molecules not only allow binding of the pathogen or toxin to the proper target cell but also appear to potentiate the toxic action. We briefly review the structure and proposed functions of cholesterol- and glycosphingolipid-rich microdomains and then describe the toxins and pathogens that interact with them. When possible the advantage conferred by the interaction with microdomains will be discussed.Abbreviation GPI glycosylphosphatidylinositol     

Association between estradiol, estrogen receptors, total lipids, triglycerides, and cholesterol in patients with benign and malignant breast tumors

This study addresses the correlation between the levels of estradiol (E₂), total lipids, triglycerides, and cholesterol in serum and tissue samples of age-matched patients with benign (40 cases; 16 were premenopausal and 24 were postmenopausal) and malignant (50 cases; 17 were premenopausal and 33 were postmenopausal) breast tumors. Estradiol levels were determined in serum and cytosol, estrogen receptors (ER) were assayed in cytosol, and total lipids, triglycerides and cholesterol were determined in serum and membrane fractions of all benign and malignant breast disease patients. Serum E₂ was significantly higher in malignant cases than benign ones (P<0.05) with a significant reduction (40%) in postmenopausal than premenopausal women. ER-positive tumors were significantly higher in postmenopausal women with malignant breast tumors than benign cases (P<0.05). Tissue levels of total lipids, triglycerides, and cholesterol were highly significantly increased in breast cancer women than women with benign breast diseases (P<0.05, P<0.005 and P<0.05 respectively) and they were also significantly correlated with estradiol levels. It could be concluded that the uptake of lipids from plasma by the tumor tissue is greatly correlated to estradiol and it may confirm the possible role of lipids as risk factor in breast cancer.     

Oxysterol profiles of normal human arteries, fatty streaks and advanced lesions.

OBJECTIVE: Human atherosclerotic lesions of different stages have quantitative differences in cholesterol and oxysterol content, but information on the oxysterol profile in fatty streaks is limited. This study aims to provide more detailed oxysterol quantification in human fatty streaks, as well as normal aorta and advanced lesions. METHODS: A newly adapted method was used, including oxysterol purification by means of a silica cartridge; and it was ensured that artifactual oxysterol formation was kept to a minimum. Cholesterol and oxysterols were estimated by GC and identification confirmed by GC-MS in samples of normal human arterial intima, intima with near-confluent fatty streaks and advanced lesions, in necropsy samples. RESULTS: The oxysterols 7 alpha-hydroxycholesterol, cholesterol-5 beta, 6 beta-epoxide, cholesterol-5 alpha, 6 alpha-epoxide, 7 beta-hydroxycholesterol, 7-ketocholesterol and 27-hydroxycholesterol (formerly known as 26-hydroxycholesterol) were found in all the lesions, but were at most very low in the normal aorta, both when related to wet weight and when related to cholesterol. Most components of the normal artery showed some cross-correlation on linear regression analysis, but cross-correlations were weaker in the fatty streaks and advanced lesions. However, in fatty streak there was a marked positive correlation between 27-hydroxycholesterol and cholesterol. CONCLUSION: The findings confirm that oxysterols are present in fatty streaks and advanced lesions and may arise from different cholesterol oxidation mechanisms, including free radical-mediated oxidation and enzymatic oxidation.     

Parameters of cholesterol metabolism in the human hepatoma cell line, Hep-G2

The human hepatoma cell line Hep-G2 has been shown to express the major enzymes of intra- and extracellular cholesterol metabolism. These include lecithin:cholesterol acyltransferase, acyl coenzyme A:cholesterol acyltransferase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and cholesterol-7 alpha-hydroxylase. Regulatory mechanisms that have been described in other hepatic systems also appear to be active in Hep-G2 cells: perturbations of cholesterol and triglyceride metabolism affected the enzyme activities and the accumulation of specific apolipoproteins in the culture media. The results indicate that studies of Hep-G2 cells may provide useful information for the elucidation of mechanisms of regulation of human hepatocyte cholesterol, lipoprotein, and biliary metabolism.     

Impact of an enriched-cholesterol diet on enzymatic cholesterol metabolism during rabbit gestation

An appropriate cholesterol homeostasis is vital for the maintenance and the optimal fetal development. The cholesterol is essential for the synthesis of progesterone and 17beta-estradiol, hormones that actively participate to sustain gestation. However, the administration of 0.2% enriched cholesterol diet (ECD) during rabbit gestation significantly increased the cholesterol blood profile (total-cholesterol, LDL, HDL, esterified-cholesterol and free-cholesterol) of dams and offspring, and induced a reduction of the offspring weight of 15% as compared to the control group. Enzymes involved in cholesterol metabolism (ACAT, HMG-CoA-reductase and cholesterol-7alpha-hydroxylase) are greatly influenced by cholesterol profile. We hypothesized that the administration of an ECD during rabbit gestation modifies the activity of those enzymes. Female rabbits (pregnant or not) were fed with a standard diet or an ECD. At term, livers (dams and offspring) and placentas were collected and ACAT, HMG-CoA-reductase and cholesterol-7alpha-hydroxylase activities were assayed. Our results demonstrate that gestation induced a reduction of ACAT activity (48.9%) in dam's liver and, an augmentation of HMG-CoA-reductase activity (142.4%) whereas it has no effect on cholesterol-7alpha-hydroxylase activity. The administration of the ECD has no additive effect on ACAT, but significantly reduced the HMG-CoA-reductase activity and cholesterol-7alpha-hydroxylase activity as compared with the pregnant control group. In placentas the ECD supplementation has an influence for HMG-CoA-reductase activity, where a 43% increased in observed. Any ACAT activity was detected in placenta and the ECD has no influence on the cholesterol-7alpha-hydroxylase activity. Whereas their offspring's liver present a reduction of ACAT and HMG-CoA-reductase activity. Gestation associated with ECD reduces significantly the HMG-CoA-reductase activity, decreasing the cholesterol synthesis, but placenta seems to compensate this effect by increasing its HMG-CoA-reductase activity.