Eicosapentaenoic Acid and Docosahexaenoic Acid Regulate Modified LDL Uptake and Macropinocytosis in Human Macrophages

There is evidence that long chain n-3 PUFA (such as from fish oils) provide atheroprotection through, in part, changes in macrophage function although it has not been fully determined whether these n-3 PUFA target cellular mechanisms that control macrophage foam cell formation. Therefore, we investigated whether the n-3 PUFA, EPA and DHA, modulate modified low-density lipoprotein (LDL) uptake by human macrophages. The uptake of fluorophore labeled acetylated LDL (AcLDL) and/or oxidized LDL (OxLDL) by THP-1 macrophages and primary human monocyte-derived macrophages were measured by flow cytometry following co-incubation with EPA or DHA in vitro. DHA inhibited both AcLDL and OxLDL uptake in human macrophages whilst EPA reduced AcLDL and increased OxLDL uptake. These effects were only partly explained by changes in the mRNA and protein expression of key scavenger receptors, such as CD36 and scavenger receptor-A, in these cells suggesting the involvement of a scavenger receptor-independent mechanism. EPA and DHA inhibited macropinocytosis, as measured by Lucifer Yellow uptake, in human macrophages and attenuated the expression of Syndecan-4, which has been implicated in the uptake of other modified forms of LDL. EPA and DHA reduced modified LDL uptake by human macrophages through a mechanism that is in part scavenger receptor-independent and may involve inhibition of macropinocytosis and Syndecan-4 expression. This suggests that both EPA and DHA are capable of regulating macrophage foam cell formation and adds to the evidence describing an atheroprotective role for n-3 PUFA, implicating them as potential therapeutic agents for the treatment of clinical atherosclerosis.     

miR-223 Inhibits Lipid Deposition and Inflammation by Suppressing Toll-Like Receptor 4 Signaling in Macrophages

Atherosclerosis and its complications rank as the leading cause of death with the hallmarks of lipid deposition and inflammatory response. MicroRNAs (miRNAs) have recently garnered increasing interests in cardiovascular disease. In this study, we investigated the function of miR-223 and the underlying mechanism in atherosclerosis. In the atherosclerotic ApoE^−/− mice models, an obvious increase of miR-223 was observed in aortic atherosclerotic lesions. In lipopolysaccharide (LPS) activated macrophages, its expression was decreased. The miR-223 overexpression significantly attenuated macrophage foam cell formation, lipid accumulation and pro-inflammatory cytokine production, which were reversed by anti-miR-223 inhibitor transfection. Mechanism assay corroborated that miR-223 negatively regulated the activation of the toll-like receptor 4 (TLR4)-nuclear factor-κB (NF-κB) pathway. Pretreatment with a specific inhibitor of NF-κB (pyrrolidinedithiocarbamate, PDTC) strikingly abrogated miR-223 silence-induced lipid deposition and inflammatory cytokine production. Furthermore, PI3K/AKT was activated by miR-223 up-regulation. Pretreatment with PI3K/AKT inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 strikingly ameliorated the inhibitory effects of miR-223 on the activation of TLR4 and p65, concomitant with the increase in lipid deposition and inflammatory cytokine production. Together, these data indicate that miR-223 up-regulation might abrogate the development of atherosclerosis by blocking TLR4 signaling through activation of the PI3K/AKT pathway, and provides a promising therapeutic avenue for the treatment of atherosclerosis.     

Dyslipidemic Diabetic Serum Increases Lipid Accumulation and Expression of Stearoyl-CoA Desaturase in Human Macrophages

Type 2 diabetes and dyslipidemia are risk factors for cardiovascular disease. However, mechanisms by which hypertriglyceridemia influences atherogenesis remain unclear. We examined effects of dyslipidemic diabetic serum on macrophage lipid accumulation as a model of foam cell formation. Normal human macrophages were cultured in media supplemented with 10% serum from non-diabetic normolipidemic or non-diabetic hypercholesterolemic adults versus adults with Type 2 diabetes; diabetes and hypertriglyceridemia; or diabetes and hypercholesterolemia. Exposure to diabetic sera resulted in increased macrophage fatty acids (2-3 fold higher, both saturated and unsaturated). Macrophage expression of CD36, scavenger receptor A (SR-A) and stearoyl-CoA desaturase (SCD) was increased, most prominently in macrophages exposed to hypertriglyceridemic diabetic serum (twofold increase in CD36 and fourfold increase in SCD, p?相似文献   

C‐reactive protein upregulates lectin‐like oxidized LDL receptor‐1 expression in THP‐1‐derived macrophages through NF‐κB

Lectin‐like oxidized LDL receptor‐1 (LOX‐1), a newly identified scavenger receptor, has been increasingly linked to atherosclerosis. C‐reactive protein (CRP), a prototypic inflammatory marker, has been proven to promote atherogenesis. In this study, we evaluated the in vitro effects of CRP on LOX‐1 expression and the associated signal transduction pathway in THP‐1‐derived macrophages. Our study showed that incubation of macrophages with CRP significantly enhanced expression of LOX‐1 protein and mRNA levels in macrophages in a dose‐dependent manner; this expression could be suppressed by the nuclear factor kappa B (NF‐κB) pathway inhibitor BAY11‐7085. However, LOX‐1 was not inhibited by the inhibitor of mitogen‐activated protein kinase (MAPK) proteins (SP600125‐JNK/SAPK, SB203580‐p38, and U0126‐ERK1/2) in macrophages. In conclusion, human native CRP up‐regulated LOX‐1 expression in THP‐1‐derived macrophages primarily through the NF‐κB signaling pathway. Practical applications: Identification of LOX‐1 and definition of its biological role in pathophysiological states provided a new clue for understanding the nature of oxLDL uptake into macrophages. Internalization of modified lipoprotein by macrophages and foam cell formation are critical events in hypertension, diabetes mellitus, and dyslipidemia, which are the most important risk factors for atherosclerosis. As a characteristic inflammatory marker, CRP has been proven to play a pivotal role in promoting atherogenesis. However, crosstalk between CRP and LOX‐1 on macrophages has not been elucidated. Therefore, determining the regulatory process for LOX‐1 and the underlying signal transduction pathways may provide a new insight into the mechanism of atherosclerosis.     

AGE-RAGE Axis Stimulates Oxidized LDL Uptake into Macrophages through Cyclin-Dependent Kinase 5-CD36 Pathway via Oxidative Stress Generation

Advanced glycation end products (AGEs) are localized in macrophage-derived foam cells within atherosclerotic lesions, which could be associated with the increased risk of atherosclerotic cardiovascular disease under diabetic conditions. Although foam cell formation of macrophages has been shown to be enhanced by AGEs, the underlying molecular mechanism remains unclear. Since cyclin-dependent kinase 5 (Cdk5) is reported to modulate inflammatory responses in macrophages, we investigated whether Cdk5 could be involved in AGE-induced CD36 gene expression and foam cell formation of macrophages. AGEs significantly increased Dil-oxidized low-density lipoprotein (ox-LDL) uptake, and Cdk5 and CD36 gene expression in U937 human macrophages, all of which were inhibited by DNA aptamer raised against RAGE (RAGE-aptamer). Cdk5 and CD36 gene expression levels were correlated with each other. An antioxidant, N-acetyl-l-cysteine, mimicked the effects of RAGE-aptamer on AGE-exposed U937 cells. A selective inhibitor of Cdk5, (R)-DRF053, attenuated the AGE-induced Dil-ox-LDL uptake and CD36 gene expression, whereas anti-CD36 antibody inhibited the Dil-ox-LDL uptake but not Cdk5 gene expression. The present study suggests that AGEs may stimulate ox-LDL uptake into macrophages through the Cdk5–CD36 pathway via RAGE-mediated oxidative stress.     

Foam Cells as Therapeutic Targets in Atherosclerosis with a Focus on the Regulatory Roles of Non-Coding RNAs

Atherosclerosis is a major cause of human cardiovascular disease, which is the leading cause of mortality around the world. Various physiological and pathological processes are involved, including chronic inflammation, dysregulation of lipid metabolism, development of an environment characterized by oxidative stress and improper immune responses. Accordingly, the expansion of novel targets for the treatment of atherosclerosis is necessary. In this study, we focus on the role of foam cells in the development of atherosclerosis. The specific therapeutic goals associated with each stage in the formation of foam cells and the development of atherosclerosis will be considered. Processing and metabolism of cholesterol in the macrophage is one of the main steps in foam cell formation. Cholesterol processing involves lipid uptake, cholesterol esterification and cholesterol efflux, which ultimately leads to cholesterol equilibrium in the macrophage. Recently, many preclinical studies have appeared concerning the role of non-encoding RNAs in the formation of atherosclerotic lesions. Non-encoding RNAs, especially microRNAs, are considered regulators of lipid metabolism by affecting the expression of genes involved in the uptake (e.g., CD36 and LOX1) esterification (ACAT1) and efflux (ABCA1, ABCG1) of cholesterol. They are also able to regulate inflammatory pathways, produce cytokines and mediate foam cell apoptosis. We have reviewed important preclinical evidence of their therapeutic targeting in atherosclerosis, with a special focus on foam cell formation.     

Lipoproteins modify the macrophage uptake of triacylglycerol emulsion and of zymosan particles by similar mechanisms

The uptake of lipids and formation of foam cells are key events in atherosclerosis and in eruptive xanthomata formation in primary hyperchylomicronemia. Here we have compared the influence of low density lipoprotein (LDL), oxidized LDL (oxLDL), high density lipoprotein (HDL), and delipidated HDL (apoHDL) on the uptake by macrophages of zymosan (an insoluble fraction of yeast cell walls) and of triglyceride-rich emulsion (EM) particles that resemble chylomicrons, but, like zymosan, are equally devoid of protein components. Zymosan internalization is known to occur through unspecific phagocytosis, whereas natural chylomicrons are taken up by several specific lipoprotein receptors. We found that phagocytosis is not promoted as much by oxLDL as by normal LDL. HDL-coated zymosan was found to be inert and apoHDL slightly enhanced phagocytosis. LDL and apoHDL promoted the uptake of EM while oxLDL and HDL significantly inhibited the uptake. Therefore, the data support that HDL, and not apoHDL, particles inhibit EM uptake. We concluded that by using lipoprotein-coated zymosan particles, we could demonstrate different biological effects of LDL, oxLDL, HDL, and apoHDL on macrophage phagocytosis and that this method could be useful to delineate components of the various lipoproteins important for the propagation or inhibition of the formation of foam cells.     

Danshensu Promotes Cholesterol Efflux in RAW264.7 Macrophages

Contemporary research suggests that macrophage foam cell and cholesterol efflux defect play pivotal role in atherogenesis. We reported on the heretofore unknown therapeutic effect of Danshensu (DSS) in reducing intracellular cholesterol level and unraveled the mechanism of DSS promotes cholesterol efflux. Oxidized low‐density lipoprotein stimulation of Raw264.7 cells into foam cells, which were treated with DSS and co‐treated with Simvastatin and Rosiglitazone. PPARγ, ABCA1, ABCG1, SR‐BI, CD36, and LXR‐α mRNA were quantified by Real‐Time PCR. Western blotting was used to determine protein expression of PPARγ, ABCA1 and CD36. Cellular cholesterol handling was studied by measurement of intracellular lipid droplets concentration and cholesterol efflux. DSS significantly reduced scavenger receptor CD36 and its orthologue SR‐BI. In addition, DSS stimulated the upregulation of cellular cholesterol exporters ABCA1 and ABCG1 to reduce intracellular lipid accumulation. DSS can reduce lipid deposition in Raw264.7 foam cells by balancing CD36 and ABCA1 protein expression.     

Gardenamide A Protects RGC-5 Cells from H2O2-Induced Oxidative Stress Insults by Activating PI3K/Akt/eNOS Signaling Pathway

Gardenamide A (GA) protects the rat retinal ganglion (RGC-5) cells against cell apoptosis induced by H₂O₂. The protective effect of GA was completely abrogated by the specific phosphoinositide 3-kinase (PI3K) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, and the specific protein kinase B (Akt) inhibitor Akt VIII respectively, indicating that the protective mechanism of GA is mediated by the PI3K/Akt signaling pathway. The specific extracellular signal-regulated kinase (ERK1/2) inhibitor PD98059 could not block the neuroprotection of GA. GA attenuated the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) induced by H₂O₂. Western blotting showed that GA promoted the phosphorylation of ERK1/2, Akt and endothelial nitric oxide synthase (eNOS), respectively, and effectively reversed the H₂O₂-inhibited phosphorylation of these three proteins. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 completely inhibited the GA-activated phosphorylation of Akt, while only partially inhibiting eNOS. This evidence implies that eNOS may be activated directly by GA. PD98059 attenuated only partially the GA-induced phosphorylation of ERK1/2 with/without the presence of H₂O₂, indicating that GA may activate ERK1/2 directly. All these results put together confirm that GA protects RGC-5 cells from H₂O₂ insults via the activation of PI3K/Akt/eNOS signaling pathway. Whether the ERK1/2 signaling pathway is involved requires further investigations.     

MicroRNA-328-5p Alleviates Macrophage Lipid Accumulation through the Histone Deacetylase 3/ATP-binding cassette transporter A1 pathway

MicroRNA-328 (miR-328) was reported to protect against atherosclerosis, but its role in foam cell formation remains unknown. The aim of this study was to investigate the effect of miR-328-5p on macrophage lipid accumulation and the underlying mechanisms. The results showed that miR-328-5p expression was robustly decreased in oxidized low-density lipoprotein (ox-LDL)-treated macrophages. Treatment of human acute monocytic leukemia cel (THP-1) macrophage-derived foam cells with a miR-328-5p mimic markedly increased [³H]-cholesterol efflux, inhibited lipid droplet accumulation, and decreased intracellular total cholesterol (TC), free cholesterol (FC) and cholesteryl ester (CE) contents. Upregulation of miR-328-5p also reduced the expression of histone deacetylase 3 (HDAC3) but increased the levels of ATP-binding cassette transporter A1 (ABCA1) in THP-1 macrophage-derived foam cells. Mechanistically, miR-328-5p inhibited HDAC3 expression by directly targeting its 3′UTR, thereby promoting ABCA1 expression and the subsequent cholesterol efflux. Furthermore, miR-328-5p mimic treatment did not affect the uptake of Dil-ox-LDL or the expression of scavenger receptor-A (SR-A), thrombospondin receptor (CD36) and ABCG1. Taken together, these findings suggest that miR-328-5p alleviates macrophage lipid accumulation through the HDAC3/ABCA1 pathway.     

Crosstalk between Delta Opioid Receptor and Nerve Growth Factor Signaling Modulates Neuroprotection and Differentiation in Rodent Cell Models

Both opioid signaling and neurotrophic factor signaling have played an important role in neuroprotection and differentiation in the nervous system. Little is known about whether the crosstalk between these two signaling pathways will affect neuroprotection and differentiation. Previously, we found that nerve growth factor (NGF) could induce expression of the delta opioid receptor gene (Oprd1, dor), mainly through PI3K/Akt/NF-κB signaling in PC12h cells. In this study, using two NGF-responsive rodent cell model systems, PC12h cells and F11 cells, we found the delta opioid neuropeptide [d-Ala², d-Leu⁵] enkephalin (DADLE)-mediated neuroprotective effect could be blocked by pharmacological reagents: the delta opioid antagonist naltrindole, PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, MAPK inhibitor PD98059, and Trk inhibitor K252a, respectively. Western blot analysis revealed that DADLE activated both the PI3K/Akt and MAPK pathways in the two cell lines. siRNA Oprd1 gene knockdown experiment showed that the upregulation of NGF mRNA level was inhibited with concomitant inhibition of the survival effects of DADLE in the both cell models. siRNA Oprd1 gene knockdown also attenuated the DADLE-mediated neurite outgrowth in PC12h cells as well as phosphorylation of MAPK and Akt in PC12h and F11 cells, respectively. These data together strongly suggest that delta opioid peptide DADLE acts through the NGF-induced functional G protein-coupled Oprd1 to provide its neuroprotective and differentiating effects at least in part by regulating survival and differentiating MAPK and PI3K/Akt signaling pathways in NGF-responsive rodent neuronal cells.     

Oxidized Low‐Density Lipoprotein Inhibits THP‐1‐Derived Macrophage Autophagy via TET2 Down‐regulation

Oxidized low‐density lipoprotein (ox‐LDL) is an independent risk factor of atherosclerosis. However, the mechanism underlying its pro‐atherosclerosis roles has not yet been well explored. DNA demethylation modification, via DNA methyltransferases or ten‐eleven‐translocation (TET) family, is a crisis epigenetic regulation for various biological and pathological processes. This study aimed to investigate the effects of ox‐LDL on macrophage autophagy and its potential epigenetic mechanism. Results showed that after treatment with 0, 10, 20, 40 or 80 mg/L ox‐LDL for 24 h, the autophagy markers Beclin 1 and LC3 expression were obviously decreased at protein levels (P < 0.05). The mRNA and protein expression of TET2 was evidently decreased (P < 0.05). After pre‐treatment with TET2 siRNA, the mRNA and protein levels of Beclin 1 and LC3 decreased compared with the 80 mg/L treatment group (P < 0.01). The mRNA and protein levels of Beclin 1 and LC3‐II were up‐regulated (P < 0.05) in the 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) of pretreatment group. Consistent with the Western blot results, cell immunofluorescence showed that the protein concentration of LC3‐II decreased in the TET2 siRNA group and increased in the 5‐aza‐2′‐deoxycytidine group. Taken together, these results showed that DNA demethylation modifications regulate ox‐LDL‐treated THP‐1 macrophages autophagy and TET2 might be a novel regulator.     

Involvement of PI3K Pathway in Glioma Cell Resistance to Temozolomide Treatment

The aim of the study was to investigate the anticancer potential of LY294002 (PI3K inhibitor) and temozolomide using glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells. Apoptosis, autophagy, necrosis, and granules in the cytoplasm were identified microscopically (fluorescence and electron microscopes). The mitochondrial membrane potential was studied by flow cytometry. The activity of caspases 3, 8, and 9 and Akt was evaluated fluorometrically, while the expression of Beclin 1, PI3K, Akt, mTOR, caspase 12, and Hsp27 was determined by immunoblotting. SiRNA was used to block Hsp27 and PI3K expression. Cell migration and localization of Hsp27 were tested with the wound healing assay and immunocytochemistry, respectively. LY294002 effectively diminished the migratory potential and increased programmed death of T98G and MOGGCCM. Autophagy was dominant in MOGGCCM, while apoptosis was dominant in T98G. LY294002 with temozolomide did not potentiate cell death but redirected autophagy toward apoptosis, which was correlated with ER stress. A similar effect was observed after blocking PI3K expression with siRNA. Transfection with Hsp27 siRNA significantly increased apoptosis related to ER stress. Our results indicate that inhibition of the PI3K/Akt/mTOR pathway sensitizes glioma cells to apoptosis upon temozolomide treatment, which was correlated with ER stress. Hsp27 increases the resistance of glioma cells to cell death upon temozolomide treatment.     

Low Amount of Salinomycin Greatly Increases Akt Activation,but Reduces Activated p70S6K Levels

The present study identified a novel salinomycin (Sal)-sensitization mechanism in cancer cells. We analyzed the signal proteins Akt, Jnk, p38, Jak, and Erk1/2 in cancer cell lines that had arrested growth following low amounts of Sal treatment. We also tested the signal molecules PI3K, PDK1, GSK3β, p70S6K, mTOR, and PTEN to analyze the PI3K/Akt/mTOR pathway. The results showed that Sal sensitization positively correlates with large reductions in p70S6K activation. Interestingly, Akt was the only signal protein to be significantly activated by Sal treatment. The Akt activation appeared to require the PI3K pathway as its activation was abolished by the PI3K inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin. The Akt activation by Sal was conserved in the other cell lines analyzed, which originated from other organs. Both Akt activation and C-PARP production were proportionally increased with increased doses of Sal. In addition, the increased levels of pAkt were not reduced over the time course of the experiment. Co-treatment with Akt inhibitors sensitized the Sal-treated cancer cells. The results thereby suggest that Akt activation is increased in cells that survive Sal treatment and resist the cytotoxic effect of Sal. Taken together; these results indicate that Akt activation may promote the resistance of cancer cells to Sal.     

BMP4 Protects Rat Pulmonary Arterial Smooth Muscle Cells from Apoptosis by PI3K/AKT/Smad1/5/8 Signaling

Bone morphogenetic protein-4 (BMP4), a member of the transforming growth factor β (TGF-β) family of growth factors, is activated and increased under hypoxic conditions, which plays an important role in the progression of pulmonary arterial hypertension (PAH). Previous studies have shown that BMP4 is involved in the regulation of proliferation, differentiation, migration and apoptosis of various cell types. However, the precise mechanisms involved in the regulation of pulmonary artery smooth muscle cells (PASMCs) in PAH are still incompletely understood. It has been reported that AKT is a critical regulator of cell survival and vascular remodeling. Therefore, there may be crosstalk between BMP4 anti-apoptotic processes and PI3K/AKT survival effect in rat PASMCs. To test this hypothesis, we performed confocal, cell viability measurement, mitochondrial potential, real-time polymerase chain reaction (PCR), and Western blot analysis to determine the role of BMP4 on cell survival and apoptosis. We found that hypoxia up-regulated the expression of BMP4. BMP4 promoted cell survival, reduced mitochondrial depolarization, and increased the expression of Bcl-2 and procaspase-3 in PASMCs under serum-deprived condition. These effects were reversed by PI3K/AKT inhibitors ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin). Thus, these findings indicate that BMP4 protects PASMCs from apoptosis at least in part, mediated via the PI3K/AKT pathway.     

Lewis y Regulate Cell Cycle Related Factors in Ovarian Carcinoma Cell RMG-I in Vitro via ERK and Akt Signaling Pathways

Objective

To investigate the effect of Lewis y overexpression on the expression of proliferation-related factors in ovarian cancer cells.

Methods

mRNA levels of cyclins, CDKs, and CKIs were measured in cells before and after transfection with the α1,2-fucosyltransferase gene by real-time PCR, and protein levels of cyclins, CDKs and CKIs were determined in cells before and after gene transfection by Western blot.

Results

Lewis y overexpression led to an increase in both mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E in ovarian cancer cells, decrease in both mRNA and protein expression levels of p16 and p21, and decrease of p27 at only the protein expression level without change in its mRNA level. There were no differences in proteins and the mRNA levels of CDK2, CDK4 and CDK6 before and after gene transfection. Anti-Lewis y antibody, ERK and PI3K pathway inhibitors PD98059 and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 reduced the difference in cyclin and CKI expression caused by Lewis y overexpression.

Conclusion

Lewis y regulates the expression of cell cycle-related factors through ERK/MAPK and PI3K/Akt signaling pathways to promote cell proliferation.     

Ubiquitin-Conjugating Enzyme 9 Promotes Epithelial Ovarian Cancer Cell Proliferation in Vitro

Epithelial ovarian cancer (EOC) is one of the leading causes of cancer deaths in women worldwide. Ubiquitin-conjugating enzyme 9 (Ubc9), the sole conjugating enzyme for sumoylation, regulates protein function and plays an important role in sumoylation-mediated cellular pathways. Although sumoylation plays a key role in DNA repair and tumorgenesis, whether Ubc9 is involved in EOC progression remains unknown. In the present study, we constructed Ubc-9 expressed recombined plasmid pEGFP-N1-Ubc9. The mRNA levels of Ubc9 were confirmed in human ovarian cell lines before and after transfection with pEGFP-N1-Ubc9 or small interfering RNA (siRNA) targeted Ubc9 by real-time polymerase chain reaction (PCR). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to observe the effect of Ubc9 on cell proliferation. The protein levels of Ubc9, and proliferation-related signals Akt and physphorylated Akt were determined by Western blot. Our results showed that proliferation of EOC cells increased significantly in Ubc9 overexpressing cells, but decreased in Ubc9 knockdown cells. The physphorylation of Akt showed similar trends. In addition, the inhibitor of PI3K/Akt signaling pathway, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, dramatically inhibited the growth of Ubc9 overexpressing cells. Therefore, Ubc9 gene plays an important role in cell proliferation in EOC through PI3K/Akt signaling pathway.     

COX-2 Inhibition and Inhibition of Cytosolic Phospholipase A2 Increase CD36 Expression and Foam Cell Formation in THP-1 Cells

Cardiovascular safety of cyclooxygenase (COX)-2-selective inhibitors and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) is of worldwide concern. COX-2 inhibitors and NSAIDs act by inhibiting arachidonic acid metabolism to prostaglandins. They confer a cardiovascular hazard manifested as an elevated risk of myocardial infarction. Mechanisms underlying these cardiovascular effects are uncertain. Here we determine whether interference with cytosolic phospholipase A2 (cPLA-2) or COX-2 through pharmacologic blockade or silencing RNA impacts expression of scavenger receptor CD36 and scavenger receptor A, both involved in cholesterol uptake in monocytes and macrophages. THP-1 human monocytes and human peripheral blood mononuclear cells were exposed to celecoxib, a COX-2 selective inhibitor currently in clinical use, and to arachidonyl trifluoromethyl ketone (AACOCF3), an arachidonic acid analog that selectively inhibits cPLA-2. Celecoxib and AACOCF3 each upregulated expression of CD36, but not scavenger receptor A, as determined by quantitative PCR and immunoblotting. Silencing of cPLA-2 or COX-2 had comparable effects to pharmacologic treatments. Oil red O staining revealed a profound increase in foam cell transformation of THP-1 macrophages exposed to either celecoxib or AACOCF3 (both 25 μM), supporting a role for the COX pathway in maintaining macrophage cholesterol homeostasis. Demonstration of disrupted cholesterol balance by AACOCF3 and celecoxib provides further evidence of the possible mechanism by which COX inhibition may promote lipid overload leading to atheromatous lesion formation and increased cardiovascular events.