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• W2028544092 abstract "The flavonoid quercetin 3-glucoside (Q3G) protected SH-SY5Y, HEK293, and MCF-7 cells against hydrogen peroxide-induced oxidative stress. cDNA microarray studies suggested that Q3G-pretreated cells subjected to oxidative stress up-regulate the expression of genes associated with lipid and cholesterol biosynthesis. Q3G pretreatment elevated both the expression and activation of sterol regulatory element-binding protein-2 (SREBP-2) only in SH-SY5Y cells subjected to oxidative stress. Inhibition of SREBP-2 expression by small interfering RNA or small molecule inhibitors of 2,3-oxidosqualene:lanosterol cyclase or HMG-CoA reductase blocked Q3G-mediated cytoprotection in SH-SY5Y cells. By contrast, Q3G did not protect either HEK293 or MCF-7 cells via this signaling pathway. Moreover, the addition of isopentenyl pyrophosphate rescued SH-SY5Y cells from the inhibitory effect of HMG-CoA reductase inhibition. Last, Q3G pretreatment enhanced the incorporation of [14C]acetate into [14C]cholesterol in SH-SY5Y cells under oxidative stress. Taken together, these studies suggest a novel mechanism for flavonoid-induced cytoprotection in SH-SY5Y cells involving SREBP-2-mediated sterol synthesis that decreases lipid peroxidation by maintaining membrane integrity in the presence of oxidative stress. The flavonoid quercetin 3-glucoside (Q3G) protected SH-SY5Y, HEK293, and MCF-7 cells against hydrogen peroxide-induced oxidative stress. cDNA microarray studies suggested that Q3G-pretreated cells subjected to oxidative stress up-regulate the expression of genes associated with lipid and cholesterol biosynthesis. Q3G pretreatment elevated both the expression and activation of sterol regulatory element-binding protein-2 (SREBP-2) only in SH-SY5Y cells subjected to oxidative stress. Inhibition of SREBP-2 expression by small interfering RNA or small molecule inhibitors of 2,3-oxidosqualene:lanosterol cyclase or HMG-CoA reductase blocked Q3G-mediated cytoprotection in SH-SY5Y cells. By contrast, Q3G did not protect either HEK293 or MCF-7 cells via this signaling pathway. Moreover, the addition of isopentenyl pyrophosphate rescued SH-SY5Y cells from the inhibitory effect of HMG-CoA reductase inhibition. Last, Q3G pretreatment enhanced the incorporation of [14C]acetate into [14C]cholesterol in SH-SY5Y cells under oxidative stress. Taken together, these studies suggest a novel mechanism for flavonoid-induced cytoprotection in SH-SY5Y cells involving SREBP-2-mediated sterol synthesis that decreases lipid peroxidation by maintaining membrane integrity in the presence of oxidative stress. Cholesterol, phospholipids, and sphingolipids are major structural components of the eukaryotic plasma membrane that play an essential role in maintaining membrane integrity. In the hydrophobic region of the membrane bilayer, sterols fill the spaces created by the acyl chains of phospholipids, thereby conferring rigidity and decreasing permeability (1Yeagle P.L. Biochim. Biophys. Acta. 1985; 822: 267-287Crossref PubMed Scopus (1261) Google Scholar). The biological function of membrane proteins is influenced by cholesterol in a number of ways (2Yeagle P.L. Biochimie (Paris). 1991; 73: 1303-1310Crossref PubMed Scopus (299) Google Scholar). Cholesterol levels can regulate the activity of enzymes involved in the biosynthetic pathway of this lipid and form membrane rafts that participate in signal transduction (3Incardona J.P. Eaton S. Curr. Opin. Cell Biol. 2000; 12: 193-203Crossref PubMed Scopus (179) Google Scholar). The importance of raft-associated cholesterol in cancer cell proliferation, migration, and cell survival has been well documented (4Zhuang L. Kim J. Adam R.M. Solomon K.R. Freeman M.R. J. Clin. Invest. 2005; 115: 959-968Crossref PubMed Scopus (446) Google Scholar). Cholesterol is derived endogenously from acetyl-CoA and exogenously by low density lipoprotein (LDL) 4The abbreviations used are: LDLlow density lipoproteinQ3Gquercetin 3-glucosideQ dihydratequercetin dihydrateMTT(3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide)SCD1stearoyl-CoA-desaturase 1HMG-CoA reductase3-hydroxy-3-methylglutaryl-coenzyme A reductaseSREBPsterol regulatory element-binding proteinSCAPSREBP cleavage-activating proteinROSreactive oxygen speciessiRNAsmall interfering RNAOSCioxidosqualene:lanosterol cyclase inhibitorLDLrLDL receptorPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assayLDHlactate dehydrogenaseTUNELterminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end in situ labelingSAMFSouthern Alberta Microarray FacilityRTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenasetBHQtert-butylhydroquinoneSAMSignificance Analysis of MicroarrayqRTquantitative RTIPPisopentenyl pyrophosphate. receptor-mediated uptake of plasma LDL (5Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4566) Google Scholar). The synthesis and uptake of cholesterol are regulated by the transcription factors sterol regulatory element-binding proteins SREBPs (6Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). Alternate splicing of SREBP-1 gives rise to SREBP-1a and SREBP-1c, which activates genes involved in fatty acid metabolism, whereas SREBP-2 activates genes critical to cholesterol synthesis (6Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). SREBP-2 is synthesized as a 125-kDa precursor protein. When cholesterol levels are low, SREBP cleavage-activating protein (SCAP) escorts SREBP-2 from the endoplasmic reticulum to Golgi, where SREBP-2 is proteolytically cleaved by proteases into a mature form (65 kDa) that translocates to the nucleus and binds to the sterol regulatory element, triggering the transcription of genes necessary for cholesterol synthesis (7Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar). If cholesterol levels exceed cellular demands, the SCAP·SREBP complex is sequestered in the endoplasmic reticulum by the insulin-induced gene product known as Insig-1 (8Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar). Recently, plasma membrane compartments rich in cholesterol have been reported to participate in cell survival pathways that reduce the injurious oxidative stress (9Yang B. Oo T.N. Rizzo V. FASEB J. 2006; 20: 1501-1503Crossref PubMed Scopus (116) Google Scholar). low density lipoprotein quercetin 3-glucoside quercetin dihydrate (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) stearoyl-CoA-desaturase 1 3-hydroxy-3-methylglutaryl-coenzyme A reductase sterol regulatory element-binding protein SREBP cleavage-activating protein reactive oxygen species small interfering RNA oxidosqualene:lanosterol cyclase inhibitor LDL receptor phosphate-buffered saline enzyme-linked immunosorbent assay lactate dehydrogenase terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end in situ labeling Southern Alberta Microarray Facility reverse transcription glyceraldehyde-3-phosphate dehydrogenase tert-butylhydroquinone Significance Analysis of Microarray quantitative RT isopentenyl pyrophosphate. Oxidative stress is a pathophysiological state that occurs when free radicals and reactive oxygen species (ROS) exceed the ability of antioxidant small molecules and proteins to neutralize them (10Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4851) Google Scholar). Oxidative stress has been implicated in a number of pathological conditions (11Ames B.N. Science. 1983; 221: 1256-1264Crossref PubMed Scopus (2746) Google Scholar, 12Halliwell B. Haemostasis. 1993; 23: 118-126PubMed Google Scholar, 13Yagi K. Chem. Phys. Lipids. 1987; 45: 337-351Crossref PubMed Scopus (1267) Google Scholar). The injurious events triggered by ROS are thought to include lipid peroxidation (14Girotti A.W. J. Lipid Res. 1998; 39: 1529-1542Abstract Full Text Full Text PDF PubMed Google Scholar), ion channel modification, DNA damage, and protein oxidation (15Gutteridge J.M. Halliwell B. Ann. N. Y. Acad. Sci. 2000; 899: 136-147Crossref PubMed Scopus (819) Google Scholar). Lipids are susceptible to oxidative damage because of their high degree of unsaturation and abundance in cell membranes. Neurons in the central nervous system are highly susceptible to oxidative stress due to their high rate of aerobic metabolism, presence of catalysts such as heavy metals that generate free radicals, excitotoxic amino acids, and low levels of antioxidants (16Montine K.S. Quinn J.F. Zhang J. Fessel J.P. Roberts L.J. Morrow J.D. Montine T.J. Chem. Phys. Lipids. 2004; 128: 117-124Crossref PubMed Scopus (218) Google Scholar, 17Halliwell B. J. Neurochem. 2006; 97: 1634-1658Crossref PubMed Scopus (2095) Google Scholar, 18Gilgun-Sherki Y. Rosenbaum Z. Melamed E. Offen D. Pharmacol. Rev. 2002; 54: 271-284Crossref PubMed Scopus (478) Google Scholar). Hence, we used the neuroblastoma SH-SY5Y cells as an in vitro model to assess the effects of oxidative stress on a neuronal-like cell line. Oxidative stress generated by free radicals is counteracted by sophisticated antioxidant defense systems (19Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3226) Google Scholar, 20Sies H. Exp. Physiol. 1997; 82: 291-295Crossref PubMed Scopus (2492) Google Scholar); however, the excessive production of ROS during pathological conditions may overwhelm endogenous antioxidant defenses resulting in tissue injury. Antioxidants derived from dietary sources have been shown to reduce oxidative tissue damage (21Youdim K.A. Spencer J.P. Schroeter H. Rice-Evans C. Biol. Chem. 2002; 383: 503-519Crossref PubMed Scopus (198) Google Scholar). Among the most potent dietary free radical scavengers identified to date are a class of polyphenolic compounds known as flavonoids found in wine, fruits, vegetables, and teas (21Youdim K.A. Spencer J.P. Schroeter H. Rice-Evans C. Biol. Chem. 2002; 383: 503-519Crossref PubMed Scopus (198) Google Scholar). Epidemiological data suggest that apple flavonoids reduce the risk of cancer, cardiovascular disease, and neurological disorders (22Boyer J. Liu R.H. Nutr. J. 2004; 3: 1-15Crossref PubMed Google Scholar). Quercetin and its glycoside derivatives are the most abundantly consumed flavonoids in the diet, reaching levels of 30–40 mg/day (23Spencer J.P. Abd-el-Mohsen M.M. Rice-Evans C. Arch. Biochem. Biophys. 2004; 423: 148-161Crossref PubMed Scopus (280) Google Scholar). Flavonoids are known to scavenge free radicals, inhibit a variety of kinases, reduce lipid peroxidation, inhibit apoptosis, prevent platelet aggregation, and exhibit anti-proliferative effects (24Williams R.J. Spencer J.P. Rice-Evans C. Free Radic. Biol. Med. 2004; 36: 838-849Crossref PubMed Scopus (1729) Google Scholar, 25Peng I.W. Kuo S.M. J. Nutr. 2003; 133: 2184-2187Crossref PubMed Scopus (74) Google Scholar, 26Molina M.F. Sanchez-Reus I. Iglesias I. Benedi J. Biol. Pharm. Bull. 2003; 26: 1398-1402Crossref PubMed Scopus (241) Google Scholar). Several flavonoids have been documented to cross the blood-brain barrier and to protect neurons from cell death in both in vitro and in vivo models of neurodegenerative diseases (27Dajas F. Rivera-Megret F. Blasina F. Arredondo F. Abin-Carriquiry J.A. Costa G. Echeverry C. Lafon L. Heizen H. Ferreira M. et al.Braz. J. Med. Biol. Res. 2003; 36: 1613-1620Crossref PubMed Scopus (192) Google Scholar, 28Mandel S. Amit T. Reznichenko L. Weinreb O. Youdim M.B. Mol. Nutr. Food Res. 2006; 50: 229-234Crossref PubMed Scopus (245) Google Scholar, 29Youdim K.A. Shukitt-Hale B. Joseph J.A. Free Radic. Biol. Med. 2004; 37: 1683-1693Crossref PubMed Scopus (320) Google Scholar). In the present study, we first demonstrate cytoprotective effects of Q3G against hydrogen peroxide injury associated with oxidative stress in SH-SY5Y cells. In order to determine if Q3G-mediated cytoprotection against oxidative stress is applicable to other cell lines that are not neuronal in origin, we also determined that Q3G protects the human embryonic kidney cell line HEK293 and the human breast cancer cell line MCF-7 from oxidative injury. Then using cDNA microarrays to profile changes in gene expression associated with Q3G-mediated cytoprotection in SH-SY5Y cells, we report that only in cells pretreated with Q3G and then subjected to oxidative stress was the expression of numerous genes implicated in cholesterol biosynthesis elevated. Since the transcriptional regulating factor SREBP-2 plays a critical role in this biosynthesis and was activated in cells pretreated with Q3G and subjected to oxidative stress, cholesterol synthesis was blocked using siRNA technology to knock down SREBP-2 expression and chemical inhibitors to block the biosynthetic enzymes HMG-CoA reductase and 2,3-oxidosqualene:lanosterol cyclase (OSC). We show using these approaches that Q3G-induced de novo cholesterol synthesis plays a pivotal role in the cytoprotective effects of this flavonoid in SH-SY-5Y cells perhaps by enhancing membrane integrity that resists lipid peroxidation in the face of oxidative stress. Materials—Q3G was purchased from ChromaDex Inc. (Santa Ana, CA). The purity of Q3G was greater than 99.0% as determined by high pressure liquid chromatography/UV, NMR, and mass spectrometry. Caspase-3-cleaved spectrin antibody was generously provided by Dr. Donald Nicholson (Merck). Oxidosqualene:lanosterol cyclase inhibitor (OSCi), designated as RO0714565 (IC50 = 10 nm), was generously provided by Hoffman-La Roche (Pharmaceutical Division, Basal, Switzerland). [14C]Acetate, sodium salt (55 mCi/mmol; 1.66–2.22 GBq/mmol), and [14C]cholesterol (58.0 mCi/mmol; 2.15 GBq/mmol) were purchased from PerkinElmer and Amersham Biosciences, respectively. Cell culture reagents were obtained from Hyclone. All other chemicals and reagents were purchased from Sigma. Plasmids—The LDLp-588luc and TK-LXRE3-luc plasmids were gifts from Dr. D. S. Ory (30Frolov A. Zielinski S.E. Crowley J.R. Dudley-Rucker N. Schaffer J.E. Ory D.S. J. Biol. Chem. 2003; 278: 25517-25525Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). LDLp-588luc contains the human LDL receptor (LDLr) promoter upstream of the luciferase reporter gene. The β-galactosidase construct was generously provided by Dr. C. Sinal (Department of Pharmacology, Dalhousie University, Halifax, Canada). Cell Culture—The human neuroblastoma cells (SH-SY5Y), human embryonic kidney cells (HEK293), and human breast cancer cells (MCF-7) were obtained from the American Type Culture Collection. SH-SY5Y, MCF-7, and HEK293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% serum (10% fetal bovine serum for SH-SY5Y and MCF-7 cells; 10% horse serum for HEK293 cells), 2 mm l-glutamine, 1 mm sodium pyruvate, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere of 5% CO2. These cell lines were seeded at an initial density of 5 × 105 cells/ml in a 75-cm2 flask and passaged every third day. SH-SY5Y cells doubled every 48 h, whereas HEK293 and MCF-7 cells doubled every 24 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay—Cell viability was determined using the MTT assay (supplemental Fig. 1, A–D). SH-SY5Y, HEK293, and MCF-7 cells were seeded in a 96-well plate at a density of 1 × 104 cells/100 μl and treated with varying concentrations of Q3G (0.01–100 μm), quercetin dihydrate (0.01–100 μm), or Me2SO vehicle for 6 or 18 h. Following a rinse in PBS, the cells were subjected to an H2O2 insult (500 μm for 15 min for SH-SY5Y cells, 500 μm for 3 h for HEK293 cells, and 800 μm for 24 h for MCF-7 cells, respectively). After several washes, the cells were maintained in the growth medium for 18 h. The cells were then incubated with 0.5 mg/ml MTT (Sigma) at 37 °C for 4 h. The formazan crystals generated by viable mitochondrial succinate dehydrogenase from MTT were extracted using an equal volume of the solubilizing buffer (0.01 n HCl and 10% SDS). Absorbance was measured at 562 nm in an ELx800uv microplate reader (Bio-tek Instruments, Inc.). The resultant data were expressed as the percentage of viable cells relative to untreated controls. Cell Death ELISA—A cell death ELISA kit that detects cytoplasmic histone-associated DNA fragments in cell lysates was used to assess cell death according to the manufacturer's instructions (Roche Applied Science). Briefly, cells were seeded in a 24-well plate at a density of 5 × 104 cells/500 μl and treated with Q3G (10 μm) or tert-butylhydroquinone (tBHQ) (5 μm) for 18 h. Following a rinse in PBS, the cells were subjected to an H2O2 insult (500 μm for 15 min). After several washes, the cells were maintained in the growth medium for 18 h. The positive control was prepared according to the manufacturer's instructions (Roche Applied Science). Absorbance was read at 405 nm using an ELx800uv microplate reader (Bio-tek Instruments). DNA fragmentation was expressed as an enrichment factor, a measure of specific enrichment of mono- and oligonucleosomes in the cell lysates. The enrichment factor was calculated as a ratio of the absorbance of the test sample to that of the untreated control. Cytotoxicity Assay—Cell membrane integrity was assayed by measuring the release of lactate dehydrogenase (LDH) using the CytoTox nonradioactive kit (Promega). A positive control was prepared according to the manufacturer's instructions (Promega). Cells were plated in a 96-well plate at a density of 1 × 104 cells/100 μl in phenol red-free Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 1 mm sodium pyruvate, and 2 mm l-glutamine. After 18 h, cells were incubated with Q3G (10 μm) for 6 h. The medium was replaced, and cells were subjected to an H2O2 insult (500 μm for 15 min). After washing, the cells were incubated with mevastatin (1 μm) for 18 h. The cells were then centrifuged at 250 × g for 4 min, and 50 μl of medium was removed from each well of the plate and transferred to another 96-well plate. An equal volume of substrate solution containing 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride salts and diaphorase was added to the medium and incubated in the dark at room temperature for 30 min. The reaction was terminated by the addition of 50 μl of stop solution to each of the wells. Absorbance was measured at 490 nm using an ELx800uv microplate reader (Bio-tek Instruments). The background value was subtracted, and the result was expressed as a percentage of LDH release compared with the positive control. Terminal Deoxynucleotidyltransferase-mediated Deoxyuridine Triphosphate Nick End in Situ Labeling (TUNEL)—TUNEL labeling was performed to detect damaged cells by labeling the nicked end of DNA with terminal deoxynucleotidyltransferase using the Apo Tag kit as per the manufacturer's instructions (Roche Applied Science) (supplemental Fig. 2A). Cells seeded on a coverslip in a 24-well plate at a density of 5 × 104 cells/500 μl were treated with Q3G (10 μm) for 18 h. Following a rinse in PBS, the cells were subjected to H2O2 insult (500 μm for 15 min). After several washes, the cells were maintained in growth medium for 18 h. We included both a positive control (cells treated with 10 units of DNase for 20 min) and a negative control (cells not treated with terminal deoxynucleotidyltransferase enzyme) in the assay. TUNEL staining was then performed, and slides were mounted with DakoCytomation fluorescent mounting medium (DakoCytomation, Carpinteria, CA). The images were captured on a Zeiss inverted microscope using a Nikon camera. Determination of ROS—ROS was measured using 5-(and 6-)carboxy-2,7-dichlorofluorescin diacetate (Molecular Probes) as substrate that is oxidized to a fluorescent product in the presence of ROS (supplemental Fig. 2B). Cells were seeded in a 24-well plate at a density of 5 × 105 cells/500 μl in phenol red free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1 mm sodium pyruvate, and 2 mm l-glutamine. Cells were then incubated with Q3G, tBHQ, or vehicle (0.01 and 0.05% Me2SO) for 18 h. Following a rinse in PBS, the cells were subjected to the H2O2 insult (500 μm for 15 min) and treated with 0.11 mg/ml horseradish peroxidase for 15 min. 5-(and 6-)Carboxy-2,7-dichlorofluorescin diacetate (10 μm) was added to the cells immediately after the insult and incubated at 37 °C for 15 min. Subsequently, the cells were washed with PBS and lysed in 10 mm Tris-HCl buffer containing 0.5% Tween 20. The lysates were centrifuged at 10,000 × g for 10 min, and the supernatant was added to an opaque 96-well plate (Costar). Fluorescence was measured using Flx800 microplate fluorescence reader (Bio-tek Instruments) with an excitation wavelength (485 nm) and an emission wavelength (528 nm). ROS was expressed as -fold induction compared with untreated cells that received no oxidative insult. cDNA Microarray Studies—cDNA microarray studies were performed by the Southern Alberta Microarray Facility (SAMF) using human 14K microarray slides (GEO Platform accession numbers GPL3963 and GPL3964), printed at the University of Calgary. These arrays contain 13,972 70-mer oligonucleotides designed against the UniGene data base (Operon version 1.0) spotted in duplicate. For further information concerning the genes represented on these microarray chips and the microarrays used in these experiments, please see the SAMF site on the World Wide Web. RNA Extraction—In the first experiment, SH-SY5Y cells were treated with Q3G (10 μm) or Me2SO vehicle (0.05%) for 6 h. In a second experiment, SH-SY5Y cells were treated with Q3G (10 μm) or Me2SO vehicle (0.05%) for 6 h and subjected to an H2O2 insult (500 μm for 15 min) and allowed to recover for 6 h in growth medium before RNA extraction. Total RNA was extracted using an RNeasy minicolumn (Qiagen). The integrity of total RNA was determined on a 1% formaldehyde-agarose gel, and the absorbance of RNA was measured at 260 and 280 nm using a spectrophotometer. RNA samples having a 260/280 absorbance ratio of 1.9–2.0 were subsequently used for microarray analysis. cDNA Synthesis, Purification, and Hybridization—cDNA microarray studies were carried out by the SAMF at the University of Calgary. cDNA labeling was performed using the Fair-Play Microarray Labeling Kit II (catalog number 252006; Stratagene). For the detailed protocol, please see the Web site). Meaurement of Microarray Data and Specification—The scans were saved as image files in TIFF format and imported into QuantArray™ version 3.0 (PerkinElmer Life Sciences) microarray analysis software used for spot identification, quantification, and background estimation. The quantified and imaged gpr files were then loaded into Gene Traffic Duo™ (Iobion) for microarray data management and analysis. The data were filtered to flag spots with intensities of less than 100 units, or less than twice the average background. The data were normalized according to the Lowess method resident in the Gene Traffic software (31Yang Y.H. Dudoit S. Luu P. Lin D.M. Peng V. Ngai J. Speed T.P. Nucleic Acids Res. 2002; 30: 1-10Crossref PubMed Scopus (39) Google Scholar). In order to identify genes that were differentially expressed and statistically significant, Significance Analysis of Microarray (SAM) version 2.2 software was used (available on the World Wide Web). The data set created in Gene Traffic 4.0 was analyzed by SAM using the following criteria: one class analysis, median center arrays, and 100 permutations. SAM plots (supplemental Fig. 3, A and B) and SAM tables were generated at corresponding Δ values. The number of significant genes with a -fold change greater than 1.5 and a false discovery rate of <7% were determined. These significant genes were further analyzed through Panther software (available on the World Wide Web) to delineate the potential biological processes involved (supplemental Fig. 3C). The pathways defined by this set of genes were analyzed using Pathway Architect (Stratagene). Quantitative Real Time PCR—Quantitative RT-PCR was performed using the DNA Engine Opticon 2 System (MJ Research) (supplemental Figs. 4, A–E, and 5, A and B). Total RNA was isolated from Q3G-, Q dihydrate-, and Me2SO-treated SH-SY5Y, HEK293, and MCF-7 cells that were subjected to oxidative stress using the RNeasy minicolumn (Qiagen). Briefly, 3 μg of DNase-treated total RNA was reverse transcribed using a First Strand cDNA synthesis kit according to the manufacturer's instructions (Superarray Incorporation Inc.). The 20-μl RT reaction mix was diluted 5-fold in RNase-free water. In order to validate up-regulated genes, cDNA from Q3G-treated cells was serially diluted 10-fold (5 points in duplicates), and the standard curve was generated for GAPDH and the genes of interest (SCD1 and HMG-CoA reductase and SREBP-2, respectively). To validate a down-regulated gene (sestrin 1), cDNA from Me2SO-treated cells was serially diluted 10-fold (5 points in duplicates), and standard curves were generated for GAPDH and sestrin 1, respectively. A negative (no RT control) was included in all of the experiments. Triplicates of control and experimental cDNA samples were included in the experiment at an appropriate dilution. PCR master mix was prepared using an RT2 Real-Time™ PCR kit using gene-specific primers in 25 μl/well of a 96-well plate as per the manufacturer's instructions (Superarray Incorporation Inc.). The cycling parameter included activation of Taq polymerase at 95 °C for 15 min. This step was followed by denaturation at 95 °C/15 s, annealing for SCD1 at 60 °C/30 s with the primers 5′-TACCGCTGGCACATCAACTT-3′ (sense) and 5′-TTGGAGACTTTCTTCCGGTCA-3′ (antisense) (32Wang J. Yu L. Schmidt R.E. Su C. Huang X. Gould K. Cao G. Biochem. Biophys. Res. Commun. 2005; 332: 735-742Crossref PubMed Scopus (169) Google Scholar) (product size, 87 bp); annealing for HMG-CoA reductase at 55 °C/30 s with the primers 5′-TACCATGTCAGGGGTACGTC-3′ (sense) and 5′-CAAGCCTAGAGACATAATCATC-3′ (antisense) (33Skarits C. Fischer S. Haas O.A. Clin. Chim. Acta. 2003; 336: 27-37Crossref PubMed Scopus (7) Google Scholar) (product size, 247 bp); annealing for sestrin 1 at 58 °C/30 s with the primers 5′-GGCAAACCATTTTGAGGAAA-3′ (sense) and 5′-ACTCCCCACTTGGAGGATCT-3′ (antisense) (PRIMER 3 software) (available on the World Wide Web) (product size, 278 bp); annealing for SREBP-2 at 62 °C/40 s with the primers 5′-CCCTTCAGTGCAACGGTCATTCAC-3′ (sense) and 5′-GATGCTCAGTGGCACTGACTCTTC-3′ (antisense), respectively (33Skarits C. Fischer S. Haas O.A. Clin. Chim. Acta. 2003; 336: 27-37Crossref PubMed Scopus (7) Google Scholar) (product size, 401 bp) and primer extension at 72 °C/30 s for a total of 40 cycles. The GAPDH Amplimer set (Clontech) was used to amplify GAPDH (450 bp). The melting curve analysis was performed to verify the accurate amplification of target amplicon. Data analysis was performed using Opticon software version 2.02. Using the standard curve generated for SCD1, HMG-CoA reductase, SREBP-2, sestrin 1, and GAPDH, respectively, the relative -fold increase in gene expression in the Q3G-treated sample over the Me2SO control was calculated using the comparative CT method (ΔΔCT) (34Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (127160) Google Scholar) and was quantified using 2-ΔΔCT with GAPDH as the internal control. The data were expressed as the relative -fold increase or decrease in gene expression compared with the Me2SO control. Lipid Peroxidation Assay—Lipid peroxidation is the method of choice for detecting phospholipid oxidation in cells either by measuring the initial products of oxidative attack, such as the lipid hydroperoxides and conjugated dienes, or by measuring the breakdown products of polyunsaturated fatty acid, namely malondialdehyde and 4-hydroxynonenal. A lipid peroxidation kit (Calbiochem) was used to measure lipid hydroperoxides generated by lipid oxidation utilizing a redox reaction with ferrous ions. The reaction of hydroperoxides with ferrous ions resulted in the generation of ferric ions that were detected using thiocyanate as a chromogen. Briefly, cells were seeded in a 6-well plate at a density of 5 × 105 cells/ml and incubated with Q3G (10 μm) or Me2SO (0.05%) for 6 h. The cells were then rinsed with PBS and subjected to H2O2 insult (500 μm for 15 min). The medium was replaced, and after 6 h, the lipid hydroperoxides were extracted from the cells using Extract R-saturated methanol and deoxygenated chloroform as per the manufacturer's instructions (Calbiochem). For the lipid peroxidation assay, a standard curve was generated. Briefly, standard and test samples were diluted in deoxygenated chloroform/methanol mixture (2:1) in a reaction volume of 950 μl in a glass tube. Chromogen substrate was freshly prepared, added to the samples (50 μl), and incubated at room temperature for 5 min. A volume of 300 μl was transferred from the glass tube to a glass 96-well plate, and absorbance was read at 490 nm using an ELx800uv microplate reader (Bio-tek Instruments). The lipid hydroperoxides in the test sample were extrapolated from a standard curve and expressed as nm/mg of protein. Cholesterol Assay—SH-SY5Y cells were seeded in a 6-well plate at a density of 5 × 105 cells/ml. Cells were incubated with Q3G (10 μm) or Me2SO vehicle (0.05%) for 6 h and exposed to H2O2 insult (500 μm for 15 min). After washing and incubation in growth medium for 6 h, the cells were counted. A phase separation method (chloroform/m" @default.
• W2028544092 created "2016-06-24" @default.
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• W2028544092 date "2008-01-01" @default.
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• W2028544092 title "Quercetin 3-Glucoside Protects Neuroblastoma (SH-SY5Y) Cells in Vitro against Oxidative Damage by Inducing Sterol Regulatory Element-binding Protein-2-mediated Cholesterol Biosynthesis" @default.
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