Lipoprotein internalisation induced by oncogenic AMPK activation is essential to maintain glioblastoma cell growth.

AIM OF THE STUDY: Metabolic adaptations are essential during tumour growth to maintain the high proliferation levels exhibited by cancer cells. In this study, we examined the transformations that occurred in the lipid metabolism in astrocytic tumours, and the possible role of the fuel-sensing enzyme AMPK. Metabolic targets might help design new and effective drugs for cancer. METHODS: To accomplish this objective, we studied both mice and human astrocytic tumours. We first used a mouse model of astrocytoma driven by oncogenic H-RasV12 and/or with PTEN deletion based on the common constitutive activation of the Raf/MEK/ERK and PI3K/AKT cascades in human astrocytomas. We then confirmed the results in human glioblastoma cell lines and in glioblastoma tissue samples from patients. RESULTS: We show that the high levels of activated AMPK, observed in astrocytic tumours, increase extracellular lipid internalisation and reduce energy expenditure by inhibiting 'de novo' fatty acid (FA) synthesis, which allows tumour cells to obtain building blocks and energy to be able to create new organelles and new cells. CONCLUSIONS: Our findings demonstrate that AMPK plays a crucial role in glioblastoma cell growth and suggest that blocking lipoprotein receptors could potentially be used as a plausible therapeutic approach for these and other type of tumours with high levels of AMPK.

Aralin, a type II ribosome-inactivating protein from Aralia elata, exhibits selective anticancer activity through the processed form of a 110-kDa high-density lipoprotein-binding protein: a promising anticancer drug.

Aralin from Aralia elata is a newly identified type II ribosome- inactivating protein, which preferentially induces apoptosis in cancer cells. In this study, we identified that the aralin receptor is a 110-kDa high-density lipoprotein-binding protein (HDLBP), which functions as a HDL receptor. The sensitivities of tumor cell lines to aralin were dependent on the expression levels of the 110-kDa HDLBP and its forced expression in aralin-resistant Huh7 cells conferred aralin sensitivity. HDLBP-knockdown HeLa cells showed a significant aralin resistance in vitro and in vivo. Conversely, ectopic expression of the 150-kDa HDLBP resulted in increased aralin sensitivity in vivo, accompanying enhanced expression of the 110-kDa HDLBP. Thus, these results showed that the 110-kDa HDLBP in lipid rafts acted as an aralin receptor and that its expression levels determined aralin sensitivity, suggesting that aralin could be a promising anticancer drug for HDLBP-overexpressing tumors.

The myeloperoxidase product hypochlorous acid generates irreversible high-density lipoprotein receptor inhibitors.

OBJECTIVE: Elevated levels of advanced oxidation protein products have been described in several chronic inflammatory diseases, like chronic renal insufficiency, rheumatoid arthritis, and atherosclerosis. Recent findings revealed that advanced oxidation protein products are inhibitors of the major high-density lipoprotein receptor, scavenger receptor class B, type 1 (SR-BI). Here, we investigated which oxidation-induced structural alterations convert plasma albumin into a high-density lipoprotein-receptor inhibitor. APPROACH AND RESULTS: Exposure of albumin to the physiological oxidant, hypochlorous acid, generated high-affinity SR-BI ligands. Protection of albumin-lysine residues before exposure to hypochlorous acid as well as regeneration of N-chloramines after oxidation of albumin completely prevented binding of oxidized albumin to SR-BI, indicating that modification of albumin-lysine residues is required to generate SR-BI ligands. Of particular interest, N-chloramines within oxidized albumin promoted irreversible binding to SR-BI, resulting in permanent receptor blockade. We observed that the SR-BI inhibitory activity of albumin isolated from chronic kidney disease patients correlated with the content of the myeloperoxidase-specific oxidation product 3-chlorotyrosine and was associated with alterations in the composition of high-density lipoprotein. CONCLUSIONS: Given that several potential atheroprotective activities of high-density lipoprotein are mediated by SR-BI, the present results raise the possibility that oxidized plasma albumin, through permanent SR-BI blockade, contributes to the pathophysiology of cardiovascular disease.

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.

Apolipoproteins D and E3 exert neurotrophic and synaptogenic effects in dorsal root ganglion cell cultures.

Co-cultures of 3T3-L1 adipocytes with neurons from the rat dorsal root ganglia (DRG) showed enhanced neuritogenesis and synaptogenesis. Microarray analysis for upregulated genes in adipocyte/DRG co-cultures currently points to apolipoproteins D and E (ApoD, ApoE) as influential proteins. We therefore tested adipocyte-secreted cholesterol and the carrier proteins ApoD and ApoE3. Cholesterol, ApoD, and ApoE3 each increased neurite outgrowth and upregulated the expression of presynaptic synaptophysin and synaptotagmin, as well as the postsynaptic density protein 95. The neurotrophic effects of ApoD and ApoE3 were associated with an increased expression of the low-density lipoprotein receptor and apolipoprotein E receptor 2. Simultaneous treatment with receptor-associated protein, an apolipoprotein receptor antagonist, inhibited the neurotrophic function of both apolipoproteins. The application of ApoD, ApoE3, and cholesterol to DRG cell cultures corresponded with increased expression of the chemokine stromal cell-derived factor 1 and its receptor CXC chemokine receptor 4 (CXCR4). Surprisingly, the inhibition of CXCR4 by the antagonistic drug AMD3100 decreased the apolipoprotein/cholesterol dependent neurotrophic effects. We thus assume that apolipoprotein-induced neuritogenesis in DRG cells interferes with CXCR4 signaling, and that adipocyte-derived apolipoproteins might be helpful in nerve repair.

Identification of the molecular target of small molecule inhibitors of HDL receptor SR-BI activity.

Scavenger receptor, class B, type I (SR-BI), controls high-density lipoprotein (HDL) metabolism by mediating cellular selective uptake of lipids from HDL without the concomitant degradation of the lipoprotein particle. We previously identified in a high-throughput chemical screen of intact cells five compounds (BLT-1-5) that inhibit SR-BI-dependent lipid transport from HDL, but do not block HDL binding to SR-BI on the cell surface. Although these BLTs are widely used to examine the diverse functions of SR-BI, their direct target(s), SR-BI itself or some other component of the SR-BI pathway, has not been identified. Here we show that SR-BI in the context of a membrane lipid environment is the target of BLT-1, -3, -4, and -5. The analysis using intact cells and an in vitro system of purified SR-BI reconstituted into liposomes was aided by information derived from structure-activity relationship (SAR) analysis of the most potent of these BLTs, the thiosemicarbazone BLT-1. We found that the sulfur atom of BLT-1 was crucially important for its inhibitory activity, because changing it to an oxygen atom resulted in the isostructural, but essentially inactive, semicarbazone derivative BLT-1sc. SAR analysis also established the importance of BLT-1's hydrophobic tail. BLTs and their corresponding inactive compounds can be used to explore the mechanism and function of SR-BI-mediated selective lipid uptake in diverse mammalian experimental models. Consequently, BLTs may help determine the therapeutic potential of SR-BI-targeted pharmaceutical drugs.

Pitavastatin inhibits remnant lipoprotein-induced macrophage foam cell formation through ApoB48 receptor-dependent mechanism.

OBJECTIVE: Atherogenic remnant lipoproteins (RLPs) are known to induce foam cell formation in macrophages in vitro and in vivo. We examined the involvement of apoB48 receptor (apoB48R), a novel receptor for RLPs, in that process in vitro and its potential regulation by pitavastatin. METHODS AND RESULTS: THP-1 macrophages were incubated in the presence of RLPs (20 mg cholesterol/dL, 24 hours) isolated from hypertriglyceridemic subjects. RLPs significantly increased intracellular cholesterol ester (CE) and triglyceride (TG) contents (4.8-fold and 5.8-fold, respectively) in the macrophages. Transfection of THP-1 macrophages with short interfering RNA (siRNA) against apoB48R significantly inhibited RLP-induced TG accumulation by 44%. When THP-1 macrophages were pretreated with pitavastatin (5 micromol/L, 24 hours), the expression of apoB48R was significantly decreased and RLP-induced TG accumulation was reduced by 56%. ApoB48R siRNA also inhibited TG accumulation in THP-1 macrophage induced by beta-very-low-density lipoprotein derived from apoE-/- mice by 58%, supporting the notion that apoB48R recognizes and takes-up RLPs in an apoE-independent manner. CONCLUSIONS: RLPs induce macrophage foam cell formation via apoB48R. Pitavastatin inhibits RLP-induced macrophage foam cell formation. The underlying mechanism involves, at least in part, inhibition of apoB48R-dependent mechanism. Our findings indicate a potential role of apoB48R in atherosclerosis. RLPs induced macrophage foam cell formation via apoB48R. Pitavastatin inhibited RLP-induced macrophage foam cell formation, at least in part, via inhibition of apoB48R expression. Our findings indicate a potential role of apoB48R in atherosclerosis.

Hepatic secretion of apoB-100 is impaired in hypobetalipoproteinemic mice with an apoB-38.9-specifying allele.

Apolipoprotein B (apoB) truncation-specifying mutations cause familial hypobetalipoproteinemia (FHBL). Lipoprotein kinetics studies have shown that production rates of apoB-100 are reduced by 70-80% in heterozygous FHBL humans, instead of the expected 50%. To develop suitable mouse models to study the underlying mechanism, apoB-38.9-only (Apob(38.9/38.9)) mice were crossbred with Apobec-1 knockout (Apobec-1(-/-)) mice or apoB-100-only (Apob(100/100)) mice to produce two lines of apoB-38.9 heterozygous mice that produce only apoB-38.9 and apoB-100, namely Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) mice. In vivo rates of apoB-100 secretion were measured using [35S]Met/Cys to label proteins and Triton WR-1339 to block apoB-100 VLDL lipolysis/uptake. Rates of secretion were reduced by 80%, rather than the expected 50%, in both Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) mice compared with those of the respective Apobec-1(-/-)/Apob(+/+) and Apob(100/100) control mice. Continuous labeling and pulse-chase experiments in primary hepatocyte cultures revealed that rates of apoB-100 synthesis by Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) hepatocytes were reduced to the expected 50% of those of the respective controls, but the efficiency of secretion of apoB-100 was significantly lower in apoB-38.9 heterozygous hepatocytes. The greater-than-expected decreases in apoB-100 production rates of FHBL heterozygous humans appear to be attributable to a defect in secretion rather than in the synthesis of apoB-100 from the unaffected apoB allele.

LDL transcytosis by protein membrane diffusion.

Endothelial cell (EC) cultures of different, selected vascular beds and/or organs were screened for receptor-mediated transport of proteins with a semipermeable filter assay. In SVEC4-10 cells, a mouse lymphoid endothelial cell line, orosomucoid, albumin, insulin and LDL were transcytosed from the apical (luminal) to basal (abluminal) side by a receptor-mediated pathway. Specific LDL transcytosis involved transport of intact LDL. A pathway of degradation of LDL and basal release involved vesicles in transport to lysosomes and amino acid merocrine secretion. This newly described transcellular passage of LDL via lysosomes, as well as the standard pathway, were reduced to 70% by PEG(50)-cholesterol (PEG-Chol). Combined results of temperature-dependence analysis and PEG(50)-cholesterol sensitivity show that two pathways contribute to general LDL transcellular passage. We suggest a mechanism of domain hopping by protein membrane diffusion of receptors as the pathway for intact LDL delivery. Based on theoretical considerations we propose that active transport by protein membrane diffusion can be facilitated by an organizational structure of lipid microdomains and polar cellular organization.

Selecting selective suppressors of selective uptake.

Scavenger receptor BI (SR-BI) is a high-density lipoprotein (HDL) receptor that mediates the selective uptake of HDL cholesteryl ester (CE) and the bidirectional flux of free cholesterol (FC). The identification of selective uptake inhibitors holds promise for mechanistic studies of SR-BI and for discovery of pharmaceuticals useful in therapy of atherosclerosis.

Advanced glycation end products (AGE) inhibit scavenger receptor class B type I-mediated reverse cholesterol transport: a new crossroad of AGE to cholesterol metabolism.

Advanced glycation end products (AGE) -modified proteins behave as active ligands for several receptors belonging to the scavenger receptor family. Scavenger receptor class B type I (SR-BI) was identified as the first high density lipoprotein (HDL) receptor that mediates selective uptake of HDL-cholesteryl esters (HDL-CE). This study investigated whether AGE proteins serve as ligands for SR-BI and affect SR-BI-mediated cholesterol transport using Chinese hamster ovary (CHO) cells overexpressing hamster SR-BI (CHO-SR-BI cells). [125I] AGE-bovine serum albumin (AGE-BSA) underwent active endocytosis and subsequent lysosomal degradation by CHO-SR-BI cells, indicating that SR-BI serves as an AGE receptor. SR-BI-mediated selective uptake of HDL-CE by CHO-SR-BI cells was efficiently inhibited by AGE-BSA although AGE-BSA had no effect on HDL binding to CHO-SR-BI cells. In addition, AGE-BSA significantly inhibited the efflux of [3H] cholesterol from CHO-SR-BI cells to HDL. These findings suggest the possibility that AGE proteins in the circulation interfere with the functions of SR-BI in reverse cholesterol transport by inhibiting the selective uptake of HDL-CE, as well as cholesterol efflux from peripheral cells to HDL, thereby accelerating diabetes-induced atherosclerosis.

Biliary anionic peptide fraction and apoA-I regulate intestinal cholesterol uptake.

Evidence is now in favor of protein-facilitated mechanisms for the intestinal cholesterol absorption. Here we report that the unesterified cholesterol uptake by rat jejunal brush border membrane vesicles (BBMVs) is efficient, saturable, and protein-mediated. The human apolipoproteins biliary anionic peptide factor (APF) and A-I (apoA-I) up-regulate micellar cholesterol uptake in a dose-dependent manner, but for all tested concentrations (0.1-20 microM), the lipid-free APF was more efficient than apoA-I. This uptake stimulation was suppressed after addition of Pabs directed to the external lipid-binding domain of the CLA-1/SR-BI and reduced by Pabs directed to the external loop of CD36. Thus, CLA-1/SR-BI and to a lesser extent CD36 are involved in the regulation of intestinal cholesterol uptake. APF, the main protein bound to biliary lipids, is likely one of their physiological effectors. As APF is an unesterified cholesterol carrier, it could facilitate the intestinal absorption of biliary cholesterol.

DAX-1 represses the high-density lipoprotein receptor through interaction with positive regulators sterol regulatory element-binding protein-1a and steroidogenic factor-1.

The high-density lipoprotein receptor (HDL-R) mediates the selective uptake of high-density lipoprotein cholesterol in nonplacental steroidogenic tissues. We have previously demonstrated that sterol regulatory element-binding protein-1a (SREBP-1a) and steroidogenic factor-1 (SF-1) positively regulate HDL-R gene transcription. In the present study, we examined whether DAX-1 (dosage-sensitive sex adrenal hypoplasia congenital critical region on the X chromosome, gene-1) could influence the expression of the HDL-R gene. Cotransfection studies demonstrated that DAX-1 was able to repress SREBP-1a and SF-1-dependent activation of the HDL-R promoter. Mammalian two-hybrid assays demonstrated that DAX-1 could interact with SREBP-1a. In addition, electrophoretic mobility shift assays demonstrated that initial incubation of DAX-1 with SREBP-1a protein in the absence of DNA prevented subsequent binding of SREBP-1a to the HDL-R sterol regulatory elements in a dose-dependent manner, whereas, in the case of SF-1, DAX-1 formed a complex with SF-1 protein on the DNA. These data suggest that DAX-1 inhibits SREBP-1a- and SF-1-dependent activation of the HDL-R promoter through different mechanisms. This investigation confirms that DAX-1 has an important role in regulating steroidogenesis by interfering with SREBP-1a and SF-1 induction of a gene involved in the transport of cholesterol, thereby limiting the amount of substrate available for steroid hormone production.

Receptor-Ck regulates giardia encystation process.

The study addressed to resolve the mechanism involved in cholesterol-dependent regulation of giardia encystation process, revealed that (a) the trophozoites have the ability to express genes coding for receptor-Ck and sterol element binding protein (SREBP); (b) inhibition of cholesterol dependent activation of receptor-Ck results in the upregulation of CWP-1 gene expression leading to encystation process. Based upon these findings, we propose that receptor-Ck dependent signalling is responsible for the regulation of giardia encystation process by cholesterol.

Identification of a receptor mediating absorption of dietary cholesterol in the intestine.

Here we show that scavenger receptor class B type I is present in the small-intestine brush border membrane where it facilitates the uptake of dietary cholesterol from either bile salt micelles or phospholipid vesicles. This receptor can also function as a port for several additional classes of lipids, including cholesteryl esters, triacylglycerols, and phospholipids. It is the first receptor demonstrated to be involved in the absorption of dietary lipids in the intestine. In liver and steroidogenic tissues, the physiological ligand of this receptor is high-density lipoprotein. We show that binding of high-density lipoprotein and apolipoprotein A-I to the brush border membrane-resident receptor inhibits uptake of cholesterol (sterol) into the brush border membrane from lipid donor particles. This finding lends further support to the conclusion that scavenger receptor BI catalyzes intestinal cholesterol uptake. Our findings suggest new therapeutic approaches for limiting the absorption of dietary cholesterol and reducing hypercholesterolemia and the risk of atherosclerosis.

Modulation of apoptosis with cytokines in B-cell chronic lymphocytic leukaemia.

In B chronic lymphocytic leukaemia (B-CLL) non-proliferating peripheral blood (PB) B cells have a long life span in vivo. In cultures, these cells die spontaneously by apoptosis. Interleukin (IL) 4 inhibits spontaneous apoptosis (SA) and promotes survival of B-CLL B cells in vitro. No such effect is observed in PB B cells from normal healthy donors. The anti-apoptotic effect of IL4 is independent of mitogen-induced cell activation but depends on the concentration of IL4. The protective effect of IL4 is specific and it is significantly reduced or abolished with anti-IL4 antibody. Interferon (IFN)-gamma and alpha- IFN also protect B-CLL B cells from apoptosis in vitro. Sera from B-CLL patients have increased levels of IFN-gamma when compared with sera from healthy donors. In addition, B-cells in B-CLL express detectable levels of IFN-gamma mRNA. Other cytokines, namely ILl, IL2, IL6 and IL7 do not affect SA of B-CLL B cells. By contrast, IL5 and antibody to apolipoprotein-1 (APO- 1) receptor increase SA significantly and in a dose-dependent manner. Interleukin 4 protects B-CLL B cells from IL5-, anti(alpha) APO-1- and steroid-induced apoptosis. The mode of action of the cytokines inducing apoptosis or protecting B-CLL B cells from dying is largely unknown. Recently the bcl-2 proto-oncogene has been associated with prolonged cell survival. However, the involvement of bel-2 in spontaneous, cytokine-induced or steroid-induced apoptosis in B-CLL has been controversial. Some authors have reported down-regulation of bcl-2 protein expression in B-CLL B-cells undergoing SA or in steroid-treated cells with IL4 preventing this down-regulation. By contrast, others observed no significant loss of bcl-2 protein expression in steroid-, alpha-APO-1 - and IL5-treated cells when compared with untreated or fresh cells. Also, no correlation between bcl-2 protein expression and protection with IL4 has been reported. In conclusion, in B-CLL IL4, IFN-gamma and alpha-IFN promote the survival of the leukaemic cells. These cytokines may therefore be involved in the pathogenesis of the B-CLL.