Article pubs.acs.org/JAFC
Lipid Rafts and Fas/FasL Pathway May Involve in Elaidic Acid-Induced Apoptosis of Human Umbilical Vein Endothelial Cells Huan Rao,† Li-Xin Ma,‡ Ting-Ting Xu,† Jing Li,† Ze-Yuan Deng,*,† Ya-Wei Fan,† and Hong-Yan Li† †
State Key Laboratory of Food Science and Technology, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330047, P.R. China ‡ Institute of Chronic and Noncommunicable Disease Control and Prevention, Shijiazhuang Center for Disease Control and Prevention, Shijiazhuang, Hebei 050019, P.R. China ABSTRACT: Our previous study showed that trans-fatty acids can cause apoptosis of endothelial cells through the caspase pathway and the mitochondrial pathway. The objective of this study was to explore how trans-fatty acids activate the caspase pathway, whether there exist specific receptors induced apoptosis by comparing normal cells and non-rafts cells treated with elaidic acid (9t18:1) and oleic acid (9c18:1), respectively. Compared to normal cells treated with 9t18:1, the cell viability increased by 13% and the number of apoptotic cells decreased by 3% in non-rafts cells treated with 9t18:1 (p < 0.05), and the expression levels of pro-apoptotic proteins such as caspase-3, -8, -9, Bax, and Bid decreased, and expression of antiapoptotic protein Bcl-2 increased (p < 0.05). In addition, Fas/FasL expression in cell membrane decreased significantly (p < 0.05). In conclusion, the lipid rafts and Fas/FasL pathway may involve in 9t18:1-induced apoptosis of human umbilical vein endothelial cells. KEYWORDS: trans-fatty acid, lipid raft, human umbilical vein endothelial cells, receptor, apoptosis
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INTRODUCTION trans-Fatty acids (TFAs) have been reported to promote vascular diseases mainly by promoting apoptosis and inflammation of vascular endothelial cells (ECs).1 Excessive apoptosis of ECs is the start of endothelial dysfunction and the development of cardiovascular disease. Epidemiological and animal studies have shown that a high intake of TFAs raise the level of serum low-density lipoprotein (LDL) and triglyceride (TG) and cause vascular endothelial injury. It can also increase expression of the soluble intercellular adhesion molecule (SICAM)-1, soluble vascular cell adhesion molecule (SVCAM)-1, and E-selectin, which are important mediators in the process of inflammation.2 TFAs induce endothelial cell apoptosis through the caspase pathway3 and promote release of endothelial inflammatory cytokines.1 However, it is unknown how the pathway is activated and whether there exist specific receptors at the cell membranes. Over the past decade, many studies have shown the existence of particular microdomains at the cell membranes: lipid rafts, which are rich in cholesterol and sphingolipids and play many important roles in cell signal transduction.4,5 Apoptosis is a genetically controlled mechanism of cell death and can be mediated by the death receptor pathway as well as the mitochondrial pathway.6 There are a number of membrane receptors and channels located in lipid rafts which promote apoptosis when activated.7 Caveolin-1 is a main protein which exhibits several functional domains in which the caveolin scaffolding domain involves in signaling pathways by the interaction with proteins.8 There have been few references about the relationship between lipid rafts and TFAs based on our knowledge. However, several studies have shown that polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and © 2013 American Chemical Society
docosahexaenoic acid (DHA), can change the component of lipid rafts.9−11 DHA was also found to change the lipid rafts regulation of interleukin receptor signaling pathway.12,13 n-3 polyunsaturated fatty acids regulated downstream signaling, activation of T cells, activation of transcription, and secretion of cytokines by changing the structure of lipid rafts, which provided evidence for n-3 polyunsaturated fatty acids in the prevention of colon cancer.14 Iwona et al.15 reported that 9c11tconjugated linoleic acid (CLA) changed cell membrane structure and then interfered with intracellular transport of epidermal growth factor receptor and its related signaling pathway, both functionally associated with lipid raft properties. Therefore, we supposed that TFAs as a kind of fatty acids might have similar connection with lipid rafts. It is interesting to know if TFAs alter vascular cell lipid rafts and stimulate cell membrane receptor proteins and activate downstream signaling pathways in the process of cell dysfunction. The aim of this study was to determine whether lipid rafts play a key role in cell dysfunction caused by TFAs and its receptor proteins activate caspase pathway.
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MATERIALS AND METHODS
Chemicals and Reagents. Dulbecco Modified Eagles Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Life Technologies (Carlsbad, USA). Anti-p21, anti-cyclin-dependent kinase (CDK) 4, anti-CDK2, anti-cyclinD1, and anticaspase-3, -8, and -9 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Anti-p53, anti-Bax, and anti-Bcl-2 antibodies were obtained from Anbo Biotechnology (California, USA). Methyl-βReceived: Revised: Accepted: Published: 798
October 28, 2013 December 23, 2013 December 23, 2013 December 23, 2013 dx.doi.org/10.1021/jf404834e | J. Agric. Food Chem. 2014, 62, 798−807
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Figure 1. The reasonable time for HUVECs treated with MβCD. (A) Effects of MβCD on HUVECs viability in different time points. HUVECs were treated with 10 mM MβCD in 10, 20, 30, 35, 40, 45, 50, 55, 60, and 65 min. Data are expressed as the mean of five individual experiments ± SD * p < 0.05. (B) The levels of cholesterol in cell membrane on different MβCD action time. Cholesterol levels were negatively correlated with the time. (C) The ultrastructure of HUVEC observed by transmission electron microscope after treatment with MβCD for 10, 60, and 90 min: (a) 10 min (magnification of 25000×), (b) 10 min (magnification of 12000×), (c) 60 min (magnification of 20000×), (d) 60 min (magnification of 12000×), (e) 90 min (magnification of 20000×), (f) 90 min (magnification of 8000×). (D) Western blot analysis of caveolin-1 before and after MβCD treatment for 50 min. cyclodextrin (MβCD) was from Aladdin (Shanghai, China). Elaidic and oleic acid (98.5% purity) were purchased from Sigma (Boston, USA). All other chemicals were of reagent grade or better. Cell Culture and TFA Treatment. Human umbilical vein endothelial cells (HUVECs, College of Medical Sciences, Nanchang University) were maintained in DMEM supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomyocin at 37 °C in a humidified atmosphere of 5% CO2 and 90% air. The cells were divided into six groups: control group (treated with 0.1 M PBS), elaidic acid group (treated with 100 μmol/L 9t18:1 for 24 h), oleic acid group (treated with 100 μmol/L 9c18:1 for 24 h), MβCD group (treated with 10 mmol/L MβCD), MβCD + elaidic acid group (incubated with 100 μmol/L9t18:1 for 24 h after treated with 10 mmol/L MβCD), and MβCD + oleic acid group (incubated with 100 μmol/L 9c18:1 for 24 h after treated with 10 mmol/L MβCD). Cytotoxicity Assay of MβCD. HUVECs in logarithm period were seeded at a density of 2 × 104−5 × 104 cells/ml into 96-well microculture plates after centrifugation and were cultured at 37 °C in humidified air with 5% CO2. HUVECs were treated with 10 mmol/L MβCD for 10, 20, 30, 40, 45, 50, 55, 60, and 65 min, respectively, and then 20 μL methyl thiazol tetrazolium (MTT) solution (5 mg/mL) was added to each well and incubated at 37 °C in humidified air with 5% CO2 for 4 h. Afterward, the medium with MTT was removed, 150 μL dimethyl sulfoxide (DMSO) was added into each well, and after
shaking the plates for about 10 min, the cell pellet was resuspended. Detection and quantification of the formazan crystals was performed by a micro plate reader (Thermo, Finland) at 490 nm. Assay of Cholesterol Efflux. To investigate the effect of 10 mmol/L MβCD on cholesterol level in cell membranes, a cholesterol assay kit (Ke Da Hong Wei, Beijing, China) was used to detect the cholesterol level in cell membranes after being treated with MβCD for different times following the instruction of the manufacturer. Cholesterol ester was decomposed into free cholesterol by cholesterol esterase, and free cholesterol was oxidized by cholesterol oxidase. Hydrogen peroxide was generated during the reaction and catalyzed by peroxidase. Chromogenic substance has a maximum absorption peak at 550 nm. Observation of HUVECs with Transmission Electron Microscope. After HUVECs were treated with 10 mmol/L MβCD for 10, 60, and 90 min, respectively. Cells were collected and fixed with 2.5% glutaraldehyde overnight. After washed in 0.1 M PBS (3 × 5 min), the cells were post fixed with 1% OsO4 for 1 h, followed by dehydration with a graded acetone series (30, 70, 90, and 2 × 100%, each for 30 min). The specimens were then embedded in resin and polymerized at 35 °C for 24 h and 60 °C for 12 h. The specimens were cut by an ultrathin microtome. The sections were collected on copper grids, stained with 0.5% lead citrate for 10 min, and rinsed with water. The 799
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Figure 2. continued
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Figure 2. Effect of TFA on apoptosis of MβCD treated or nontreated HUVECs. (A) The effect of 9t18:1 and 9c18:1 on viability of MβCD treated and nontreated HUVECs. HUVECs were treated or nontreated with MβCD for 50 min and then cultured with 9t18:1 or 9c18:1 for 24 h. (B) Apoptosis detection by flow cytometry. Cells were treated as (A) labeled with PI and Annexin V-FITC and then were analyzed by flow cytometry. The histograms represent the data obtained by flow cytometry. In upper quadrants: right, percentage of cells labeled with PI; left, percentage of cells labeled with PI and Annexin V. In lower quadrants: right, percentage of cells labeled with Annexin V; left, percentage of viable cells. (C) Western blot analysis of caspase-3, -8, -9, Bcl-2, Bax, and Bid in different groups. (D) Cytochrome C release rate with different treatments detected by ELISA kit. Data are expressed as the mean of 3−5 individual experiments ± SD * p < 0.05, ** p < 0.01, compared with the control group. # p < 0.05, ## p < 0.01, 9t18:1 group compared with the MβCD + 9t18:1 group, 9c18:1 group compared with the MβCD + 9c18:1 group. 490 nm using a micro plate reader (Spectra max190, Molecular Devices, California). Apoptosis Detection by Flow Cytometry. Cells were collected from each group and 100 μL of binding buffer added to suspend the cells. The cells were then collected by centrifugation and washed twice with PBS. Then 5 μL of fluorescein isothiocyanate (FITC) and 5 μL of propidium iodide (PI) were added to the mixture and incubated in the
images of these sections were made by transmission electron microscope. Cell Viability Assay. Briefly, at the end of the incubation, the media were removed and MTT added in each well for 4 h incubation. Then DMSO was added and shaken gently for 10 min so that the precipitation was completely dissolved. Absorbance was recorded at 801
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Figure 3. Western blot analysis of p53, p21, p27, CDK4, CyclinD1, and CDK2. Data are expressed as the mean of three individual experiments ± SD * p < 0.05, ** p < 0.01, compared with the control group. # p < 0.05, ## p < 0.01, 9t18:1 group compared with the MβCD + 9t18:1 group, 9c18:1 group compared with the MβCD + 9c18:1 group. Determination of Fatty Acids by Gas Chromatography. Total lipids were extracted from 4−6 × 107 cells using chloroform/methanol (1:1, v:v). Fatty acids were then methylated using sodium methoxide and analyzed by a gas chromatograph equipped with a flame ionization detector (model 6890 N, Agilent Technologies, Shanghai, China). Peaks were identified by their retention times relative to standards FAME mixture (no. 463). Statistical Analysis. Data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Student’s ttest using SPSS.15.0 software. p < 0.05 was indicative of significant difference.
dark for 10−15 min at room temperature. Apoptosis of the cells was detected by flow cytometry within 1 h after the addition of 500 μL of binding buffer. Western Blot Analysis of Apoptosis-Related Proteins. Total cell lysates were extracted by RIPA buffer on ice. After centrifugation at 20000g for 15 min, supernatant was taken and stored at −70 °C until use. Protein (80 μg) was mixed with loading buffer and denatured by boiling for 5 min. The proteins were separated by SDSPAGE and then transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk, membranes were then incubated with anticaspase-3, -8, and -9, anti-Bcl2, anti-Bax, and anti-Bid antibodies overnight at 4 °C, followed by incubation with horseradish peroxidaseconjugated secondary antibodies at room temperature for 1 h. Finally, protein bands were visualized by enhanced chemiluminescence (ECL) detection system (Kang Wei, Beijing, China). Immunofluorescence of Fas. Briefly, cells were fixed with 4% paraformaldehyde for 15 min and 0.5% Triton X-100 in PBS for 20 min. Cells were blocked with goat serum for 1 h and then incubated overnight at 4 °C with anti-Fas antibody (1:100). Subsequently, cells were incubated with goat antirabbit FITC antibody (1:200) for 1 h at room temperature and incubated with 40, 6-diamidino-2-phenylindole (DAPI), to label nuclei, for 5 min in a dark space. Images were acquired with a fluorescence microscope (Olympus, Japan). Measurement of Cytochrome C Level. Treated cells were washed with cold PBS twice and homogenized in cold lysis buffer containing 1 mmol/L DTT and 0.5 mmol/L PMSF on ice for 40 min and then centrifuged at 10000 rpm at 4 °C for 2 min. The supernatants were used for the measurement of cytochrome C using ELISA kit (Senxiong Biotechnology, Shanghai, China).
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RESULTS Treatment Time of MβCD in HUVECs. MβCD is a timedependent reagent which could extract cholesterol of cell and perturb the function of lipid rafts. The reasonable treatment time of MβCD (10 mmol/L) in HUVECs without cytotoxicity should be determined. As shown in Figure 1 A, cell viability decreased by 30% within 65 min treatment. Cholesterol levels (Figure 1B) dropped to 60% after treatment with MβCD for 1 h. Although the longer the treatment time with MβCD and the better extract efficiency of cholesterol, cells membranes were more seriously damaged with the increased treatment time. As shown in Figure 1C, there was almost no injury to the cells when the treatment times were less than 60 min, but obvious damages were found after 60 min, such as morphological changes, cytoplasm leakage, and increased cavities. To ensure the normal physiological conditions of cells, the reasonable 802
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Figure 4. (A) The expression of Fas and its ligand (FasL). Data are expressed as the mean of three individual experiments ± SD * p < 0.05, ** p < 0.01, compared with the control group. # p < 0.05, ## p < 0.01, 9t18:1 group compared with the MβCD + 9t18:1 group, 9c18:1 group compared with the MβCD + 9c18:1 group. (B) Visualization of Fas in HUVECs. Micrographs show the Fas (light green), nucleus (light blue), and the overlapping of images (merge) of the studied cells.
treatment time of MβCD should be within 60 min. We also examined the effect of MβCD on lipid rafts in 50 min by determining the expression of cavelin-1, one of important makers of lipid rafts. A significant decrease in cavelin-1 expression was observed (Figure 1D). The results suggest that MβCD may interrupt the function of lipid rafts. Effect of MβCD on TFA-Induced Apoptosis of HUVECs. MTT assay and flow cytometry were performed to examine the effect of TFA on cell viability and apoptosis of HUVECs. The cell viability rate in MβCD + elaidic acid group increased significantly (from 70% to 84%), and the MβCD + oleic acid
group had no obvious changes (Figure 2A). In the MβCD treated groups, the number of apoptotic cells which incubated with 9t18:1 dropped significantly by nearly 3% (the early apoptosis decreased from 5.11% to 4.50% and late apoptosis dropped to 1.5%), while the apoptotic cells of 9c18:1 group slightly increased when compared with non-MβCD treated group (Figure 2B). To determine whether lipid rafts play an important role in cell apoptosis after TFA supplementation, we compared the expression levels of apoptosis-related proteins between MβCD treated and nontreated cells. In the MβCD + elaidic acid group, 803
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Table 1. Effect of Different Treatments on Fatty Acid Composition of HUVECsa % of total fatty acids fatty acids in lipid fractions
control
∑SFA ∑cisMUFA ∑PUFA ∑TFA 9t18:1 9c18:1 ∑n-3 PUFA ∑n-6 PUFA
± ± ± ± ± ± ± ±
31.58 54.29 13.60 0.53 0.30 36.29 7.54 5.89
MβCD c
0.12 0.12b 0.00b 0.01a 0.01a 0.27b 0.03b 0.06b
33.34 53.59 12.45 0.62 0.37 36.23 6.79 5.67
± ± ± ± ± ± ± ±
elaidic acid c
4.76 3.00b 1.78b 0.02a 0.03a 0.95b 1.06b 0.72b
21.13 42.14 8.52 28.21 27.50 26.25 4.48 3.76
± ± ± ± ± ± ± ±
a
0.20 0.06a 0.36a 0.62b 0.67b 0.11a 0.34a 0.06a
MβCD + elaidic acid
oleic acid
± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ±
19.03 42.99 9.00 28.98 28.29 27.09 4.37 4.27
a
0.72 0.47a 0.35a 0.84b 0.88b 0.23a 0.24a 0.64a
23.97 66.36 9.16 0.51 0.38 50.72 4.72 4.16
MβCD + oleic acid b
0.33 0.41c 0.01a 0.07a 0.02a 0.21bc 0.05a 0.08a
24.82 64.48 10.24 0.45 0.35 48.12 5.00 4.93
± ± ± ± ± ± ± ±
0.07b 1.17c 1.24a 0.01a 0.01a 1.44c 0.05a 1.21ab
Mean values ± SD (n = 3.. Values in the same row with different letters for each species show significant differences (p < 0.05. ∑SFA, total saturated fatty acids; ∑cis MUFA, total cis monounsaturated fatty acids; ∑trans, total trans fatty acids; ∑PUFA, total polyunsaturated fatty acids; ∑n-3 PUFA, total n-3 polyunsaturated fatty acids; ∑n-6 PUFA, total n-6 polyunsaturated fatty acids.
a
were no significant differences between control and MβCD groups or between elaidic acid and MβCD + elaidic acid groups. Only did 9c18:1 level decrease in the MβCD + oleic acid group compared with the oleic acid group. The results indicated that lipid rafts had little effect on elaidic acid absorption but a slight effect on oleic acid absorption.
the expression of caspase-3, -8, -9, and Bax was down-regulated and Bcl-2 was up-regulated when compared with elaidic acid group and control group (Figure 2C). Furthermore, the release of cytochrome C from the mitochondria to cytosol decreased (Figure 2D), perhaps due to the down-regulated expression of Bid. The expression levels of pro-apoptotic proteins decreased significantly in MβCD + oleic acid group compared with control group, while expression levels of some proteins including Bax, Bcl-2, and caspase-9 almost unchanged between the MβCD + oleic acid group and the oleic acid group (Figure 2C). These results indicate that lipid rafts had greater impact on the elaidic acid group than on the oleic acid group. Effect of MβCD on TFA-Induced Cell Cycle Changes in HUVECs. As shown in Figure 3, in non-MβCD treated groups, elaidic acid significantly increased the expression of p53, p21, and p27 in comparison with other groups. The expression levels of p21 and p27 also increased in 9c18:1 group compared with control group. It has been reported that p21 and p27 could induce cell cycle arrest in G1 phase and G2/M phase through inhibition of CDK4-cyclin D1 and CDK2-cyclin E activity (18, 19). However, the expression of CDK4 and cyclin D1 increased significantly (p < 0.05) and CDK2 expression did not change significantly (p > 0.05). In MβCD treated HUVECs, 9t18:1 supplementation resulted in a down-regulation of p53, p21, p27, and CDK4-cyclin D1 compared with 9t18:1 group. It should be pointed out that expression of p53 decreased significantly compared with control group, but CDK2 had no significant changes compared with other groups. Similar data were obtained with 9c18:1. Effect of MβCD on TFA-Induced Expression of Apoptosis Receptors. As shown in Figure 4A, Fas and TNFR1, two apoptosis receptors located in lipid rafts, were investigated. The expressions of Fas and its ligand (FasL) were significantly increased in the elaidic acid group and unchanged in the oleic acid group, while they returned to normal levels in the MβCD + elaidic acid group and slightly increased in the MβCD + oleic acid group. The expression of TNFR1 had no significant change in each group (p < 0.05). As shown in Figure 4B, both Fas and its ligand (FasL) were highly expressed on the cell membranes of 9t18:1 stimulated HUVECs The death domain with each other together formed tripolymers and activated the apoptosis pathway. Effect of MβCD on Absorption of Fatty Acids in HUVECs. The levels of 9t18:1 in elaidic acid group and 9c18:1 in the oleic acid group significantly increased in comparison with control group (p < 0.05, Table 1). The results showed that the cells had obvious absorption of fatty acids. However, there
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DISCUSSION Lipid Rafts Could Involve in Elaidic Acid-Induced Apoptosis of HUVECs. TFAs resulted in systemic inflammation, insulin resistance, and adiposity resistance. Both randomized trials and observational studies demonstrated that TFAs consumption led to endothelial dysfunction.16 Previous studies have shown that several dietary fatty acids had an effect on both cell membranes and gene transcription.17,18 Qiu et al.3 showed that TFAs led to endothelial cell apoptosis through the caspase pathway and the mitochondria. However, they did not report how TFAs activated the caspase pathway. It remains unknown whether there are specific receptor-induced apoptosis or what roles cell membranes play during this process. Apoptosis was a genetically controlled mechanism of cell death with a number of specific biochemical and morphological changes, including membrane blebbing, chromatin condensation, cytoplasmic condensation, and internucleosomal fragmentation of DNA. It could be mediated by death receptor pathway as well as the mitochondria pathway.19 Lipid rafts were specific membrane microdomains which were rich in cholesterols and sphingolipids.4 Lipid rafts appeared to be involved in many biological events such as synthetic traffic, cell skeletal reconstruction, and signal transduction.5 MβCD was the most common drug that interferes with lipid rafts; its mechanism of action was to remove or disturb the specificity of membrane cholesterol. Treatment with 10 mmol/L of MβCD within 60 min did not cause significant changes in both morphology (Figure 1C) and physiology (Figure 1A) of HUVECs. The content of cholesterol and expression level of cavelin-1 in cell membrane decreased significantly (Figure 1B,D). These results indicated that treatment with 10 mmol/L of MβCD within 60 min successfully destroy lipid rafts on cell membrane. The 9t18:1 treatment decreased cell viability, increased apoptosis, and resulted in down-regulation of Bcl-2 and up-regulation of the pro-apoptotic proteins such as Bax, Bid, caspase-3, caspase-8, and caspase-9. However, when lipid rafts of the endothelial cells were destroyed, the expression levels of these pro-apoptotic proteins were decreased and antiapoptotic protein (Bcl-2) was increased in 9t18:1-induced apoptosis. These results have for 804
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the first time demonstrated that lipid rafts could influence on 9t18:1-induced apoptosis of HUVECs. Taken together, we have for the first time shown that fatty acids especially TFAs act on HUVECs by affecting lipid rafts. However, whether apoptotic receptors were destroyed or fatty acids were less absorbed remained unclear. Therefore, we conducted fatty acids composition analysis in the study. It was found that there was no significant difference in the level of fatty acids between elaidic acid group and MβCD + elaidic acid group, but the level of 9c18:1 in MβCD + oleic acid group decreased significantly (p < 0.05). We proposed that the absorption of 9t18:1 was not affected by lipid rafts, and the apoptotic receptor was the key point of apoptosis. The decreased absorption of 9c18:1 in MβCD + oleic acid group might be due to the decline of fatty acid translocase, a receptor localized in lipid rafts and nonraft plasma membrane, with an ability to help the uptake of required long-chain fatty acids for normal biological function of cells.20 Fas/FasL Pathway May Involve in 9t18:1-Induced Apoptosis of HUVECs. Two apoptosis receptors, Fas and tumor necrosis factor receptor (TNFR) 1, were located in lipid rafts. Fas (also known as CD95 or APO-1) was a type I glycosylated 45 kD membrane protein that belonged to the TNF family and transmitted a suicide signal to the cell upon binding of its ligand (FasL), a highly conserved, 40 kD glycoprotein.21 TNFR1 was also a kind of apoptotic receptor.22 Fas/FasL system was a critical signal pathway involved in apoptosis regulation in several cell types23 and could be activated by many factors, such as drugs,24 oxidative stress,25 and other stimuli. Loss of integrity of lipid rafts by drugs could diminish CD95-mediated caspase-8 activation and apoptosis.26,27 The activation of CD95 linked with its translocation into the lipid rafts. Lang et al.28 reported that CD95 signaling was triggered by the interaction between active/inactive CD95 species and lipid rafts. A number of studies have demonstrated the importance of formation and internalization of CD95containing raft in CD95-induced apoptosis of different kinds of human cells.29,30 Zhang et al.31 using a reconstituted system, found that the Fas/FasL interaction between membranes creates submicrometer membrane microdomains, and the cells then reveal various apoptosis features. Treatment of elaidic acid increased the apoptosis of HUVECs. The result of immunofluorescence analysis (Figure 4B) showed that Fas clustered on lipid rafts when HUVECs were stimulated by 9t18:1. However, the expression levels of Fas and FasL significantly decreased in nonrafts cells after 9t18:1 treatment where cell apoptosis was inhibited. These results indicated that the Fas/FasL pathway might be involved in elaidic acid-induced apoptosis of HUVECs. In conclusion, we proposed a possible molecular mechanism involved in 9t18:1-induced apoptosis and cell-cycle arrest in HUVECs (Figure 5). Lipid rafts and Fas/FasL pathway could play an important role in 9t18:1-induced apoptosis. Fas receptor and its ligand clustered in the lipid raft and then started the apoptosis program after 9t18:1 stimulated HUVECs. Then caspase-8 was activated when the signal was received that the apoptosis receptor (Fas/FasL) transmitted. The high expression of caspase-8 could increase the expression of Bid and Bax and decrease the expression of Bcl-2 and lead to release of mitochondrial cytochrome C. The mitochondrial cytochrome C which released to the cytoplasm would activate caspase-9 and then activate its downstream protein caspase-3. In addition, p53 as a critical pro-apoptotic protein and cell cycle
Figure 5. The proposed molecular mechanisms involved in 9t18:1induced apoptosis and cell-cycle arrest in HUVECs.
protein was highly expressed after being treated with 9t18:1. The high expression of p53 increased the caspase-9 and other pro-apoptotic protein like Bax. p53 could also activate the expression of p21 and then interfere with the cell cycle. 9t18:1 Affects G1/S Phase in Cell Cycle. Cells in the long term evolution have developed detection mechanisms to ensure correct DNA replication and cell cycle distribution, and these critical control stages were often called cell cycle checkpoints or restriction points. There were three major cell cycle checkpoints, namely G1/S phase, G2/M phase, and the spindle assembly checkpoint. In the process of cell cycle regulation, the control point of G1/S phase was the key point of signal transmission and integration into the nucleus and the process of cell proliferation. The human tumor suppressor protein p53 was critical for the transcriptional activation of a series of proteins involved in cell cycle control and apoptosis. p53 played its control on the cell cycle primarily through the G1/S checkpoint 32 and also regulated the G2/M checkpoint.33 Activation of p53 in response to a variety of stimuli was associated with an increase in its protein levels and post-translational modifications, which induced the activation of a number of genes like p21 and p27, leading to cell cycle arrest.34 CDK4 as a checkpoint regulates cell cycle in G1/S phase. In general, increased expression of p21 could enhance the inhibitory effect on CDKs and weaken CDK4-cyclinD1 regulation in the G1/S phase, resulting in G1 arrest and incomplete cell mitosis.35 Compared to the control, 9t18:1 induced significant increases in the expression levels of p53, p21, and p27 (p < 0.05) and the expression level of CDK4-cyclinD1 increased in 9t18:1 group. When nonraft cells were exposed to 9t18:1, p53, p21, and p27 protein levels decreased and CDK4-cyclinD1 level also decreased compared with that in the 9t18:1 group, but they were still higher than that of the control. Whether this phenomenon is the result of self-regulation or of other mechanisms needs to be further investigated.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +86 791 88304402. Funding
This study was supported by a National Natural Science Foundation of China, the Research Program of State Key Laboratory of Food Science and Technology (SKLF-TS805
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(16) Micha, R.; Mozaffarian, D. Trans fatty acids: effects on cardiometabolic health and implications for policy. Prostaglandins, Leukotrienes Essent. Fatty Acids 2008, 79, 147−152. (17) Feller, S. E.; Gawrisch, K. Properties of docosahexaenoic-acidcontaining lipids and their influence on the function of rhodopsin. Curr. Opin. Struct. Biol. 2005, 15, 416−422. (18) Roach, C.; Feller, S. E.; Ward, J. A.; Shaikh, S. R.; Zerouga, M.; Stillwell, W. Comparison of cis and trans fatty acid containing phosphatidylcholines on membrane properties. Biochemistry 2004, 43, 6344−6351. (19) Hengartner, M. O. The biochemistry of apoptosis. Nature 2000, 407, 770−776. (20) Eyre, N. S.; Cleland, L. G.; Tandon, N. N.; Mayrhofer, G. Importance of the carboxyl terminus of FAT/CD36 for plasma membrane localization and function in long-chain fatty acid uptake. J. Lipid Res. 2007, 48, 528−542. (21) Rao-Bindal, K.; Zhou, Z.; Kleinerman, E. S. MS-275 sensitizes osteosarcoma cells to Fas ligand-induced cell death by increasing the localization of Fas in membrane lipid rafts. Cell Death Dis. 2012, 3, e369. (22) Poggi, M.; Kara, I.; Brunel, J.-M.; Landrier, J.-F.; Govers, R.; Bonardo, B.; Fluhrer, R.; Haass, C.; Alessi, M. C.; Peiretti, F. Palmitoylation of TNF alpha is involved in the regulation of TNF receptor 1 signalling. Biochim. Biophys. Acta 2013, 1833, 602−612. (23) Walczak, H.; Krammer, P. H. The CD95 APO-1/Fas and the TRAIL APO-2L apoptosis systems. Exp. Cell Res. 2000, 256, 58−66. (24) Liu, W.-H.; Chang, L.-S. Fas/FasL-dependent and -independent activation of caspase-8 in doxorubicin-treated human breast cancer MCF-7 cells: ADAM10 down-regulation activates Fas/FasL signaling pathway. Int. J. Biochem. Cell Biol. 2011, 43, 1708−1719. (25) Anathy, V.; Roberson, E.; Cunniff, B.; Nolin, J. D.; Hoffman, S.; Spiess, P.; Guala, A. S.; Lahue, K. G.; Goldman, D.; Flemer, S. Oxidative processing of latent Fas in the endoplasmic reticulum controls the strength of apoptosis. Mol. Cell. Biol. 2012, 32, 3464− 3478. (26) Grassmé, H.; Cremesti, A.; Kolesnick, R.; Gulbins, E. Ceramidemediated clustering is required for CD95-DISC formation. Oncogene 2003, 22, 5457−5470. (27) Grassmé, H.; Schwarz, H.; Gulbins, E. Molecular mechanisms of ceramide-mediated CD95 clustering. Biochem. Biophys. Res. Commun. 2001, 284, 1016−1030. (28) Lang, I.; Fick, A.; Schäfer, V.; Giner, T.; Siegmund, D.; Wajant, H. Signaling active CD95 receptor molecules trigger co-translocation of inactive CD95 molecules into lipid rafts. J. Biol. Chem. 2012, 287, 24026−24042. (29) Eramo, A.; Sargiacomo, M.; Ricci-Vitiani, L.; Todaro, M.; Stassi, G.; Messina, C. G.; Parolini, I.; Lotti, F.; Sette, G.; Peschle, C. CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells. Eur. J. Immunol. 2004, 34, 1930−1940. (30) Gajate, C.; Mollinedo, F. The antitumor ether lipid ET-18OCH3 induces apoptosis through translocation and capping of Fas/ CD95 into membrane rafts in human leukemic cells. Blood 2001, 98, 3860−3863. (31) Zhang, L.; Kaizuka, Y.; Hanagata, N. Imaging of Fas−FasL membrane microdomains during apoptosis in a reconstituted cell−cell junction. Biochem. Biophys. Res. Commun. 2012, 422, 298−304. (32) Kano, H.; Arakawa, Y.; Takahashi, J. A.; Nozaki, K.; Kawabata, Y.; Takatsuka, K.; Kageyama, R.; Ueba, T.; Hashimoto, N. Overexpression of RFT induces G1-S arrest and apoptosis via p53/ p21Waf1 pathway in glioma cell. Biochem. Biophys. Res. Commun. 2004, 317, 902−908. (33) Ikeda, M.; Okamoto, I.; Tamura, K.; Satoh, T.; Yonesaka, K.; Fukuoka, M.; Nakagawa, K. Down-regulation of survivin by ultraviolet C radiation is dependent on p53 and results in G2-M arrest in A549 cells. Cancer Lett. 2007, 248, 292−298. (34) Lavin, Ma.; Gueven, N. The complexity of p53 stabilization and activation. Cell Death Differ. 2006, 13, 941−950.
200921), Ph.D. Subject Fund from Education Department (201136 011200 04), Natural Science Fund of Jiangxi Province (20114BAB2 14016). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Deming Gong for editing the manuscript. ABBREVIATIONS USED FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HDL, high-density lipoprotein; HUVECs, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule; LDL, low-density lipoprotein; MβCD, methyl-β-cyclodextrin; MTT, methyl thiazol tetrazolium; PI, propidium iodide; TFAs, trans-fatty acids
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REFERENCES
(1) Mozaffarian, D. Trans fatty acidseffects on systemic inflammation and endothelial function. Atheroscler. Suppl. 2006, 7, 29−32. (2) Ascherio, A. Trans fatty acids and blood lipids. Atheroscler. Suppl. 2006, 7, 25−27. (3) Qiu, B.; Hu, J. N.; Liu, R.; Fan, Y. W.; Li, J.; Li, Y.; Deng, Z. Y. Caspase pathway of elaidic acid 9t-C18:1-induced apoptosis in human umbilical vein endothelial cells. Cell Biol. Int. 2012, 36, 255−260. (4) Calder, P. C.; Yaqoob, P. Lipid RaftsComposition, Characterization, and Controversies. J. Nutr. 2007, 37, 545−547. (5) George, K. S.; Wu, S. Lipid raft: a floating island of death or survival. Toxicol. Appl. Pharmacol. 2012, 259, 311−319. (6) Lee, Y.; Gustafsson, Å. B. Role of apoptosis in cardiovascular disease. Apoptosis 2009, 14, 536−548. (7) Lemaire-Ewing, S.; Lagrost, L.; Néel, D. Lipid rafts: A signaling platform linking lipoprotein metabolism to atherogenesis. Atherosclerosis 2012, 221, 303−310. (8) Xu, Y.; Buikema, H.; Gilst, W. H.; Henning, R. H. Caveolae and endothelial dysfunction: filling the caves in cardiovascular disease. Eur. J. Pharmacol. 2008, 585, 256−260. (9) Stulnig, T. M.; Huber, J.; Leitinger, N.; Imre, E.-M.; Angelisová, P.; Nowotny, P.; Waldhäusl, W. Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition. J. Biol. Chem. 2001, 276, 37335−37340. (10) Li, Q.; Wang, M.; Tan, L.; Wang, C.; Ma, J.; Li, N.; Li, Y.; Xu, G.; Li, J. Docosahexaenoic acid changes lipid composition and interleukin-2 receptor signaling in membrane rafts. J. Lipid Res. 2005, 46, 1904−1913. (11) Chen, W.; Jump, D. B.; Esselman, W. J.; Busik, J. V. Inhibition of cytokine signaling in human retinal endothelial cells through modification of caveolae/lipid rafts by docosahexaenoic acid. Invest. Ophthalmol. Visual Sci. 2007, 48, 18−26. (12) Kim, W.; Fan, Y.-Y.; Barhoumi, R.; Smith, R.; McMurray, D. N.; Chapkin, R. S. n-3 Polyunsaturated fatty acids suppress the localization and activation of signaling proteins at the immunological synapse in murine CD4+ T cells by affecting lipid raft formation. J Immunol. 2008, 181, 6236−6243. (13) Fan, Y.-Y.; Ly, L. H.; Barhoumi, R.; McMurray, D. N.; Chapkin, R. S. Dietary docosahexaenoic acid suppresses T cell protein kinase Cθ lipid raft recruitment and IL-2 production. J. Immunol. 2004, 173, 6151−6160. (14) Turk, H. F.; Chapkin, R. S. Membrane lipid raft organization is uniquely modified by n-3 polyunsaturated fatty acids. Prostaglandins, Leukotrienes Essent. Fatty Acids 2013, 88, 43−47. (15) Grądzka, I.; Sochanowicz, B.; Brzóska, K.; Wójciuk, G.; Sommer, S.; Wojewódzka, M.; Gasińska, A.; Degen, C.; Jahreis, G.; Szumiel, I. cis-9,trans-11-Conjugated linoleic acid affects lipid raft composition and sensitizes human colorectal adenocarcinoma HT-29 cells to Xradiation. Biochim. Biophys. Acta 2013, 1830, 2233−2242. 806
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Lipid Rafts and Fas/FasL Pathway May Involve in Elaidic Acid-Induced Apoptosis of Human Umbilical Vein Endothelial Cells Huan Rao,† Li-Xin Ma,‡ Ting-Ting Xu,† Jing Li,† Ze-Yuan Deng,*,† Ya-Wei Fan,† and Hong-Yan Li† †
State Key Laboratory of Food Science and Technology, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330047, P.R. China ‡ Institute of Chronic and Noncommunicable Disease Control and Prevention, Shijiazhuang Center for Disease Control and Prevention, Shijiazhuang, Hebei 050019, P.R. China ABSTRACT: Our previous study showed that trans-fatty acids can cause apoptosis of endothelial cells through the caspase pathway and the mitochondrial pathway. The objective of this study was to explore how trans-fatty acids activate the caspase pathway, whether there exist specific receptors induced apoptosis by comparing normal cells and non-rafts cells treated with elaidic acid (9t18:1) and oleic acid (9c18:1), respectively. Compared to normal cells treated with 9t18:1, the cell viability increased by 13% and the number of apoptotic cells decreased by 3% in non-rafts cells treated with 9t18:1 (p < 0.05), and the expression levels of pro-apoptotic proteins such as caspase-3, -8, -9, Bax, and Bid decreased, and expression of antiapoptotic protein Bcl-2 increased (p < 0.05). In addition, Fas/FasL expression in cell membrane decreased significantly (p < 0.05). In conclusion, the lipid rafts and Fas/FasL pathway may involve in 9t18:1-induced apoptosis of human umbilical vein endothelial cells. KEYWORDS: trans-fatty acid, lipid raft, human umbilical vein endothelial cells, receptor, apoptosis
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INTRODUCTION trans-Fatty acids (TFAs) have been reported to promote vascular diseases mainly by promoting apoptosis and inflammation of vascular endothelial cells (ECs).1 Excessive apoptosis of ECs is the start of endothelial dysfunction and the development of cardiovascular disease. Epidemiological and animal studies have shown that a high intake of TFAs raise the level of serum low-density lipoprotein (LDL) and triglyceride (TG) and cause vascular endothelial injury. It can also increase expression of the soluble intercellular adhesion molecule (SICAM)-1, soluble vascular cell adhesion molecule (SVCAM)-1, and E-selectin, which are important mediators in the process of inflammation.2 TFAs induce endothelial cell apoptosis through the caspase pathway3 and promote release of endothelial inflammatory cytokines.1 However, it is unknown how the pathway is activated and whether there exist specific receptors at the cell membranes. Over the past decade, many studies have shown the existence of particular microdomains at the cell membranes: lipid rafts, which are rich in cholesterol and sphingolipids and play many important roles in cell signal transduction.4,5 Apoptosis is a genetically controlled mechanism of cell death and can be mediated by the death receptor pathway as well as the mitochondrial pathway.6 There are a number of membrane receptors and channels located in lipid rafts which promote apoptosis when activated.7 Caveolin-1 is a main protein which exhibits several functional domains in which the caveolin scaffolding domain involves in signaling pathways by the interaction with proteins.8 There have been few references about the relationship between lipid rafts and TFAs based on our knowledge. However, several studies have shown that polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and © 2013 American Chemical Society
docosahexaenoic acid (DHA), can change the component of lipid rafts.9−11 DHA was also found to change the lipid rafts regulation of interleukin receptor signaling pathway.12,13 n-3 polyunsaturated fatty acids regulated downstream signaling, activation of T cells, activation of transcription, and secretion of cytokines by changing the structure of lipid rafts, which provided evidence for n-3 polyunsaturated fatty acids in the prevention of colon cancer.14 Iwona et al.15 reported that 9c11tconjugated linoleic acid (CLA) changed cell membrane structure and then interfered with intracellular transport of epidermal growth factor receptor and its related signaling pathway, both functionally associated with lipid raft properties. Therefore, we supposed that TFAs as a kind of fatty acids might have similar connection with lipid rafts. It is interesting to know if TFAs alter vascular cell lipid rafts and stimulate cell membrane receptor proteins and activate downstream signaling pathways in the process of cell dysfunction. The aim of this study was to determine whether lipid rafts play a key role in cell dysfunction caused by TFAs and its receptor proteins activate caspase pathway.
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MATERIALS AND METHODS
Chemicals and Reagents. Dulbecco Modified Eagles Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Life Technologies (Carlsbad, USA). Anti-p21, anti-cyclin-dependent kinase (CDK) 4, anti-CDK2, anti-cyclinD1, and anticaspase-3, -8, and -9 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Anti-p53, anti-Bax, and anti-Bcl-2 antibodies were obtained from Anbo Biotechnology (California, USA). Methyl-βReceived: Revised: Accepted: Published: 798
October 28, 2013 December 23, 2013 December 23, 2013 December 23, 2013 dx.doi.org/10.1021/jf404834e | J. Agric. Food Chem. 2014, 62, 798−807
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Figure 1. The reasonable time for HUVECs treated with MβCD. (A) Effects of MβCD on HUVECs viability in different time points. HUVECs were treated with 10 mM MβCD in 10, 20, 30, 35, 40, 45, 50, 55, 60, and 65 min. Data are expressed as the mean of five individual experiments ± SD * p < 0.05. (B) The levels of cholesterol in cell membrane on different MβCD action time. Cholesterol levels were negatively correlated with the time. (C) The ultrastructure of HUVEC observed by transmission electron microscope after treatment with MβCD for 10, 60, and 90 min: (a) 10 min (magnification of 25000×), (b) 10 min (magnification of 12000×), (c) 60 min (magnification of 20000×), (d) 60 min (magnification of 12000×), (e) 90 min (magnification of 20000×), (f) 90 min (magnification of 8000×). (D) Western blot analysis of caveolin-1 before and after MβCD treatment for 50 min. cyclodextrin (MβCD) was from Aladdin (Shanghai, China). Elaidic and oleic acid (98.5% purity) were purchased from Sigma (Boston, USA). All other chemicals were of reagent grade or better. Cell Culture and TFA Treatment. Human umbilical vein endothelial cells (HUVECs, College of Medical Sciences, Nanchang University) were maintained in DMEM supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomyocin at 37 °C in a humidified atmosphere of 5% CO2 and 90% air. The cells were divided into six groups: control group (treated with 0.1 M PBS), elaidic acid group (treated with 100 μmol/L 9t18:1 for 24 h), oleic acid group (treated with 100 μmol/L 9c18:1 for 24 h), MβCD group (treated with 10 mmol/L MβCD), MβCD + elaidic acid group (incubated with 100 μmol/L9t18:1 for 24 h after treated with 10 mmol/L MβCD), and MβCD + oleic acid group (incubated with 100 μmol/L 9c18:1 for 24 h after treated with 10 mmol/L MβCD). Cytotoxicity Assay of MβCD. HUVECs in logarithm period were seeded at a density of 2 × 104−5 × 104 cells/ml into 96-well microculture plates after centrifugation and were cultured at 37 °C in humidified air with 5% CO2. HUVECs were treated with 10 mmol/L MβCD for 10, 20, 30, 40, 45, 50, 55, 60, and 65 min, respectively, and then 20 μL methyl thiazol tetrazolium (MTT) solution (5 mg/mL) was added to each well and incubated at 37 °C in humidified air with 5% CO2 for 4 h. Afterward, the medium with MTT was removed, 150 μL dimethyl sulfoxide (DMSO) was added into each well, and after
shaking the plates for about 10 min, the cell pellet was resuspended. Detection and quantification of the formazan crystals was performed by a micro plate reader (Thermo, Finland) at 490 nm. Assay of Cholesterol Efflux. To investigate the effect of 10 mmol/L MβCD on cholesterol level in cell membranes, a cholesterol assay kit (Ke Da Hong Wei, Beijing, China) was used to detect the cholesterol level in cell membranes after being treated with MβCD for different times following the instruction of the manufacturer. Cholesterol ester was decomposed into free cholesterol by cholesterol esterase, and free cholesterol was oxidized by cholesterol oxidase. Hydrogen peroxide was generated during the reaction and catalyzed by peroxidase. Chromogenic substance has a maximum absorption peak at 550 nm. Observation of HUVECs with Transmission Electron Microscope. After HUVECs were treated with 10 mmol/L MβCD for 10, 60, and 90 min, respectively. Cells were collected and fixed with 2.5% glutaraldehyde overnight. After washed in 0.1 M PBS (3 × 5 min), the cells were post fixed with 1% OsO4 for 1 h, followed by dehydration with a graded acetone series (30, 70, 90, and 2 × 100%, each for 30 min). The specimens were then embedded in resin and polymerized at 35 °C for 24 h and 60 °C for 12 h. The specimens were cut by an ultrathin microtome. The sections were collected on copper grids, stained with 0.5% lead citrate for 10 min, and rinsed with water. The 799
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Figure 2. continued
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Figure 2. Effect of TFA on apoptosis of MβCD treated or nontreated HUVECs. (A) The effect of 9t18:1 and 9c18:1 on viability of MβCD treated and nontreated HUVECs. HUVECs were treated or nontreated with MβCD for 50 min and then cultured with 9t18:1 or 9c18:1 for 24 h. (B) Apoptosis detection by flow cytometry. Cells were treated as (A) labeled with PI and Annexin V-FITC and then were analyzed by flow cytometry. The histograms represent the data obtained by flow cytometry. In upper quadrants: right, percentage of cells labeled with PI; left, percentage of cells labeled with PI and Annexin V. In lower quadrants: right, percentage of cells labeled with Annexin V; left, percentage of viable cells. (C) Western blot analysis of caspase-3, -8, -9, Bcl-2, Bax, and Bid in different groups. (D) Cytochrome C release rate with different treatments detected by ELISA kit. Data are expressed as the mean of 3−5 individual experiments ± SD * p < 0.05, ** p < 0.01, compared with the control group. # p < 0.05, ## p < 0.01, 9t18:1 group compared with the MβCD + 9t18:1 group, 9c18:1 group compared with the MβCD + 9c18:1 group. 490 nm using a micro plate reader (Spectra max190, Molecular Devices, California). Apoptosis Detection by Flow Cytometry. Cells were collected from each group and 100 μL of binding buffer added to suspend the cells. The cells were then collected by centrifugation and washed twice with PBS. Then 5 μL of fluorescein isothiocyanate (FITC) and 5 μL of propidium iodide (PI) were added to the mixture and incubated in the
images of these sections were made by transmission electron microscope. Cell Viability Assay. Briefly, at the end of the incubation, the media were removed and MTT added in each well for 4 h incubation. Then DMSO was added and shaken gently for 10 min so that the precipitation was completely dissolved. Absorbance was recorded at 801
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Figure 3. Western blot analysis of p53, p21, p27, CDK4, CyclinD1, and CDK2. Data are expressed as the mean of three individual experiments ± SD * p < 0.05, ** p < 0.01, compared with the control group. # p < 0.05, ## p < 0.01, 9t18:1 group compared with the MβCD + 9t18:1 group, 9c18:1 group compared with the MβCD + 9c18:1 group. Determination of Fatty Acids by Gas Chromatography. Total lipids were extracted from 4−6 × 107 cells using chloroform/methanol (1:1, v:v). Fatty acids were then methylated using sodium methoxide and analyzed by a gas chromatograph equipped with a flame ionization detector (model 6890 N, Agilent Technologies, Shanghai, China). Peaks were identified by their retention times relative to standards FAME mixture (no. 463). Statistical Analysis. Data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Student’s ttest using SPSS.15.0 software. p < 0.05 was indicative of significant difference.
dark for 10−15 min at room temperature. Apoptosis of the cells was detected by flow cytometry within 1 h after the addition of 500 μL of binding buffer. Western Blot Analysis of Apoptosis-Related Proteins. Total cell lysates were extracted by RIPA buffer on ice. After centrifugation at 20000g for 15 min, supernatant was taken and stored at −70 °C until use. Protein (80 μg) was mixed with loading buffer and denatured by boiling for 5 min. The proteins were separated by SDSPAGE and then transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk, membranes were then incubated with anticaspase-3, -8, and -9, anti-Bcl2, anti-Bax, and anti-Bid antibodies overnight at 4 °C, followed by incubation with horseradish peroxidaseconjugated secondary antibodies at room temperature for 1 h. Finally, protein bands were visualized by enhanced chemiluminescence (ECL) detection system (Kang Wei, Beijing, China). Immunofluorescence of Fas. Briefly, cells were fixed with 4% paraformaldehyde for 15 min and 0.5% Triton X-100 in PBS for 20 min. Cells were blocked with goat serum for 1 h and then incubated overnight at 4 °C with anti-Fas antibody (1:100). Subsequently, cells were incubated with goat antirabbit FITC antibody (1:200) for 1 h at room temperature and incubated with 40, 6-diamidino-2-phenylindole (DAPI), to label nuclei, for 5 min in a dark space. Images were acquired with a fluorescence microscope (Olympus, Japan). Measurement of Cytochrome C Level. Treated cells were washed with cold PBS twice and homogenized in cold lysis buffer containing 1 mmol/L DTT and 0.5 mmol/L PMSF on ice for 40 min and then centrifuged at 10000 rpm at 4 °C for 2 min. The supernatants were used for the measurement of cytochrome C using ELISA kit (Senxiong Biotechnology, Shanghai, China).
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RESULTS Treatment Time of MβCD in HUVECs. MβCD is a timedependent reagent which could extract cholesterol of cell and perturb the function of lipid rafts. The reasonable treatment time of MβCD (10 mmol/L) in HUVECs without cytotoxicity should be determined. As shown in Figure 1 A, cell viability decreased by 30% within 65 min treatment. Cholesterol levels (Figure 1B) dropped to 60% after treatment with MβCD for 1 h. Although the longer the treatment time with MβCD and the better extract efficiency of cholesterol, cells membranes were more seriously damaged with the increased treatment time. As shown in Figure 1C, there was almost no injury to the cells when the treatment times were less than 60 min, but obvious damages were found after 60 min, such as morphological changes, cytoplasm leakage, and increased cavities. To ensure the normal physiological conditions of cells, the reasonable 802
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Figure 4. (A) The expression of Fas and its ligand (FasL). Data are expressed as the mean of three individual experiments ± SD * p < 0.05, ** p < 0.01, compared with the control group. # p < 0.05, ## p < 0.01, 9t18:1 group compared with the MβCD + 9t18:1 group, 9c18:1 group compared with the MβCD + 9c18:1 group. (B) Visualization of Fas in HUVECs. Micrographs show the Fas (light green), nucleus (light blue), and the overlapping of images (merge) of the studied cells.
treatment time of MβCD should be within 60 min. We also examined the effect of MβCD on lipid rafts in 50 min by determining the expression of cavelin-1, one of important makers of lipid rafts. A significant decrease in cavelin-1 expression was observed (Figure 1D). The results suggest that MβCD may interrupt the function of lipid rafts. Effect of MβCD on TFA-Induced Apoptosis of HUVECs. MTT assay and flow cytometry were performed to examine the effect of TFA on cell viability and apoptosis of HUVECs. The cell viability rate in MβCD + elaidic acid group increased significantly (from 70% to 84%), and the MβCD + oleic acid
group had no obvious changes (Figure 2A). In the MβCD treated groups, the number of apoptotic cells which incubated with 9t18:1 dropped significantly by nearly 3% (the early apoptosis decreased from 5.11% to 4.50% and late apoptosis dropped to 1.5%), while the apoptotic cells of 9c18:1 group slightly increased when compared with non-MβCD treated group (Figure 2B). To determine whether lipid rafts play an important role in cell apoptosis after TFA supplementation, we compared the expression levels of apoptosis-related proteins between MβCD treated and nontreated cells. In the MβCD + elaidic acid group, 803
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Table 1. Effect of Different Treatments on Fatty Acid Composition of HUVECsa % of total fatty acids fatty acids in lipid fractions
control
∑SFA ∑cisMUFA ∑PUFA ∑TFA 9t18:1 9c18:1 ∑n-3 PUFA ∑n-6 PUFA
± ± ± ± ± ± ± ±
31.58 54.29 13.60 0.53 0.30 36.29 7.54 5.89
MβCD c
0.12 0.12b 0.00b 0.01a 0.01a 0.27b 0.03b 0.06b
33.34 53.59 12.45 0.62 0.37 36.23 6.79 5.67
± ± ± ± ± ± ± ±
elaidic acid c
4.76 3.00b 1.78b 0.02a 0.03a 0.95b 1.06b 0.72b
21.13 42.14 8.52 28.21 27.50 26.25 4.48 3.76
± ± ± ± ± ± ± ±
a
0.20 0.06a 0.36a 0.62b 0.67b 0.11a 0.34a 0.06a
MβCD + elaidic acid
oleic acid
± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ±
19.03 42.99 9.00 28.98 28.29 27.09 4.37 4.27
a
0.72 0.47a 0.35a 0.84b 0.88b 0.23a 0.24a 0.64a
23.97 66.36 9.16 0.51 0.38 50.72 4.72 4.16
MβCD + oleic acid b
0.33 0.41c 0.01a 0.07a 0.02a 0.21bc 0.05a 0.08a
24.82 64.48 10.24 0.45 0.35 48.12 5.00 4.93
± ± ± ± ± ± ± ±
0.07b 1.17c 1.24a 0.01a 0.01a 1.44c 0.05a 1.21ab
Mean values ± SD (n = 3.. Values in the same row with different letters for each species show significant differences (p < 0.05. ∑SFA, total saturated fatty acids; ∑cis MUFA, total cis monounsaturated fatty acids; ∑trans, total trans fatty acids; ∑PUFA, total polyunsaturated fatty acids; ∑n-3 PUFA, total n-3 polyunsaturated fatty acids; ∑n-6 PUFA, total n-6 polyunsaturated fatty acids.
a
were no significant differences between control and MβCD groups or between elaidic acid and MβCD + elaidic acid groups. Only did 9c18:1 level decrease in the MβCD + oleic acid group compared with the oleic acid group. The results indicated that lipid rafts had little effect on elaidic acid absorption but a slight effect on oleic acid absorption.
the expression of caspase-3, -8, -9, and Bax was down-regulated and Bcl-2 was up-regulated when compared with elaidic acid group and control group (Figure 2C). Furthermore, the release of cytochrome C from the mitochondria to cytosol decreased (Figure 2D), perhaps due to the down-regulated expression of Bid. The expression levels of pro-apoptotic proteins decreased significantly in MβCD + oleic acid group compared with control group, while expression levels of some proteins including Bax, Bcl-2, and caspase-9 almost unchanged between the MβCD + oleic acid group and the oleic acid group (Figure 2C). These results indicate that lipid rafts had greater impact on the elaidic acid group than on the oleic acid group. Effect of MβCD on TFA-Induced Cell Cycle Changes in HUVECs. As shown in Figure 3, in non-MβCD treated groups, elaidic acid significantly increased the expression of p53, p21, and p27 in comparison with other groups. The expression levels of p21 and p27 also increased in 9c18:1 group compared with control group. It has been reported that p21 and p27 could induce cell cycle arrest in G1 phase and G2/M phase through inhibition of CDK4-cyclin D1 and CDK2-cyclin E activity (18, 19). However, the expression of CDK4 and cyclin D1 increased significantly (p < 0.05) and CDK2 expression did not change significantly (p > 0.05). In MβCD treated HUVECs, 9t18:1 supplementation resulted in a down-regulation of p53, p21, p27, and CDK4-cyclin D1 compared with 9t18:1 group. It should be pointed out that expression of p53 decreased significantly compared with control group, but CDK2 had no significant changes compared with other groups. Similar data were obtained with 9c18:1. Effect of MβCD on TFA-Induced Expression of Apoptosis Receptors. As shown in Figure 4A, Fas and TNFR1, two apoptosis receptors located in lipid rafts, were investigated. The expressions of Fas and its ligand (FasL) were significantly increased in the elaidic acid group and unchanged in the oleic acid group, while they returned to normal levels in the MβCD + elaidic acid group and slightly increased in the MβCD + oleic acid group. The expression of TNFR1 had no significant change in each group (p < 0.05). As shown in Figure 4B, both Fas and its ligand (FasL) were highly expressed on the cell membranes of 9t18:1 stimulated HUVECs The death domain with each other together formed tripolymers and activated the apoptosis pathway. Effect of MβCD on Absorption of Fatty Acids in HUVECs. The levels of 9t18:1 in elaidic acid group and 9c18:1 in the oleic acid group significantly increased in comparison with control group (p < 0.05, Table 1). The results showed that the cells had obvious absorption of fatty acids. However, there
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DISCUSSION Lipid Rafts Could Involve in Elaidic Acid-Induced Apoptosis of HUVECs. TFAs resulted in systemic inflammation, insulin resistance, and adiposity resistance. Both randomized trials and observational studies demonstrated that TFAs consumption led to endothelial dysfunction.16 Previous studies have shown that several dietary fatty acids had an effect on both cell membranes and gene transcription.17,18 Qiu et al.3 showed that TFAs led to endothelial cell apoptosis through the caspase pathway and the mitochondria. However, they did not report how TFAs activated the caspase pathway. It remains unknown whether there are specific receptor-induced apoptosis or what roles cell membranes play during this process. Apoptosis was a genetically controlled mechanism of cell death with a number of specific biochemical and morphological changes, including membrane blebbing, chromatin condensation, cytoplasmic condensation, and internucleosomal fragmentation of DNA. It could be mediated by death receptor pathway as well as the mitochondria pathway.19 Lipid rafts were specific membrane microdomains which were rich in cholesterols and sphingolipids.4 Lipid rafts appeared to be involved in many biological events such as synthetic traffic, cell skeletal reconstruction, and signal transduction.5 MβCD was the most common drug that interferes with lipid rafts; its mechanism of action was to remove or disturb the specificity of membrane cholesterol. Treatment with 10 mmol/L of MβCD within 60 min did not cause significant changes in both morphology (Figure 1C) and physiology (Figure 1A) of HUVECs. The content of cholesterol and expression level of cavelin-1 in cell membrane decreased significantly (Figure 1B,D). These results indicated that treatment with 10 mmol/L of MβCD within 60 min successfully destroy lipid rafts on cell membrane. The 9t18:1 treatment decreased cell viability, increased apoptosis, and resulted in down-regulation of Bcl-2 and up-regulation of the pro-apoptotic proteins such as Bax, Bid, caspase-3, caspase-8, and caspase-9. However, when lipid rafts of the endothelial cells were destroyed, the expression levels of these pro-apoptotic proteins were decreased and antiapoptotic protein (Bcl-2) was increased in 9t18:1-induced apoptosis. These results have for 804
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the first time demonstrated that lipid rafts could influence on 9t18:1-induced apoptosis of HUVECs. Taken together, we have for the first time shown that fatty acids especially TFAs act on HUVECs by affecting lipid rafts. However, whether apoptotic receptors were destroyed or fatty acids were less absorbed remained unclear. Therefore, we conducted fatty acids composition analysis in the study. It was found that there was no significant difference in the level of fatty acids between elaidic acid group and MβCD + elaidic acid group, but the level of 9c18:1 in MβCD + oleic acid group decreased significantly (p < 0.05). We proposed that the absorption of 9t18:1 was not affected by lipid rafts, and the apoptotic receptor was the key point of apoptosis. The decreased absorption of 9c18:1 in MβCD + oleic acid group might be due to the decline of fatty acid translocase, a receptor localized in lipid rafts and nonraft plasma membrane, with an ability to help the uptake of required long-chain fatty acids for normal biological function of cells.20 Fas/FasL Pathway May Involve in 9t18:1-Induced Apoptosis of HUVECs. Two apoptosis receptors, Fas and tumor necrosis factor receptor (TNFR) 1, were located in lipid rafts. Fas (also known as CD95 or APO-1) was a type I glycosylated 45 kD membrane protein that belonged to the TNF family and transmitted a suicide signal to the cell upon binding of its ligand (FasL), a highly conserved, 40 kD glycoprotein.21 TNFR1 was also a kind of apoptotic receptor.22 Fas/FasL system was a critical signal pathway involved in apoptosis regulation in several cell types23 and could be activated by many factors, such as drugs,24 oxidative stress,25 and other stimuli. Loss of integrity of lipid rafts by drugs could diminish CD95-mediated caspase-8 activation and apoptosis.26,27 The activation of CD95 linked with its translocation into the lipid rafts. Lang et al.28 reported that CD95 signaling was triggered by the interaction between active/inactive CD95 species and lipid rafts. A number of studies have demonstrated the importance of formation and internalization of CD95containing raft in CD95-induced apoptosis of different kinds of human cells.29,30 Zhang et al.31 using a reconstituted system, found that the Fas/FasL interaction between membranes creates submicrometer membrane microdomains, and the cells then reveal various apoptosis features. Treatment of elaidic acid increased the apoptosis of HUVECs. The result of immunofluorescence analysis (Figure 4B) showed that Fas clustered on lipid rafts when HUVECs were stimulated by 9t18:1. However, the expression levels of Fas and FasL significantly decreased in nonrafts cells after 9t18:1 treatment where cell apoptosis was inhibited. These results indicated that the Fas/FasL pathway might be involved in elaidic acid-induced apoptosis of HUVECs. In conclusion, we proposed a possible molecular mechanism involved in 9t18:1-induced apoptosis and cell-cycle arrest in HUVECs (Figure 5). Lipid rafts and Fas/FasL pathway could play an important role in 9t18:1-induced apoptosis. Fas receptor and its ligand clustered in the lipid raft and then started the apoptosis program after 9t18:1 stimulated HUVECs. Then caspase-8 was activated when the signal was received that the apoptosis receptor (Fas/FasL) transmitted. The high expression of caspase-8 could increase the expression of Bid and Bax and decrease the expression of Bcl-2 and lead to release of mitochondrial cytochrome C. The mitochondrial cytochrome C which released to the cytoplasm would activate caspase-9 and then activate its downstream protein caspase-3. In addition, p53 as a critical pro-apoptotic protein and cell cycle
Figure 5. The proposed molecular mechanisms involved in 9t18:1induced apoptosis and cell-cycle arrest in HUVECs.
protein was highly expressed after being treated with 9t18:1. The high expression of p53 increased the caspase-9 and other pro-apoptotic protein like Bax. p53 could also activate the expression of p21 and then interfere with the cell cycle. 9t18:1 Affects G1/S Phase in Cell Cycle. Cells in the long term evolution have developed detection mechanisms to ensure correct DNA replication and cell cycle distribution, and these critical control stages were often called cell cycle checkpoints or restriction points. There were three major cell cycle checkpoints, namely G1/S phase, G2/M phase, and the spindle assembly checkpoint. In the process of cell cycle regulation, the control point of G1/S phase was the key point of signal transmission and integration into the nucleus and the process of cell proliferation. The human tumor suppressor protein p53 was critical for the transcriptional activation of a series of proteins involved in cell cycle control and apoptosis. p53 played its control on the cell cycle primarily through the G1/S checkpoint 32 and also regulated the G2/M checkpoint.33 Activation of p53 in response to a variety of stimuli was associated with an increase in its protein levels and post-translational modifications, which induced the activation of a number of genes like p21 and p27, leading to cell cycle arrest.34 CDK4 as a checkpoint regulates cell cycle in G1/S phase. In general, increased expression of p21 could enhance the inhibitory effect on CDKs and weaken CDK4-cyclinD1 regulation in the G1/S phase, resulting in G1 arrest and incomplete cell mitosis.35 Compared to the control, 9t18:1 induced significant increases in the expression levels of p53, p21, and p27 (p < 0.05) and the expression level of CDK4-cyclinD1 increased in 9t18:1 group. When nonraft cells were exposed to 9t18:1, p53, p21, and p27 protein levels decreased and CDK4-cyclinD1 level also decreased compared with that in the 9t18:1 group, but they were still higher than that of the control. Whether this phenomenon is the result of self-regulation or of other mechanisms needs to be further investigated.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Fax: +86 791 88304402. Funding
This study was supported by a National Natural Science Foundation of China, the Research Program of State Key Laboratory of Food Science and Technology (SKLF-TS805
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200921), Ph.D. Subject Fund from Education Department (201136 011200 04), Natural Science Fund of Jiangxi Province (20114BAB2 14016). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Deming Gong for editing the manuscript. ABBREVIATIONS USED FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HDL, high-density lipoprotein; HUVECs, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule; LDL, low-density lipoprotein; MβCD, methyl-β-cyclodextrin; MTT, methyl thiazol tetrazolium; PI, propidium iodide; TFAs, trans-fatty acids
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