Study of pharmacokinetics of prenylflavonoids and dynamics of estrogen action in sera following ingestion of epimedium using validated, ultra sensitive cell based bioassays

CHAPTER MATERIALS AND METHODS 2.1 Reagents 49 2.2 Cell lines and cell culture 53 2.3 ERα and ERβ cell-based bioassays 54 2.3.1 Establishment of ERα and ERβ stable cells 54 2.3.2 Optimization of ERα and ERβ bioassay parameters 55 2.3.3 Validation of stable ERα- and ERβ-driven reporter gene bioassays 56 2.3.4 Assay procedure for stably transfected ERα- and ERβ-responsive cell lines 57 2.3.5 Bioassay calibration curves 58 2.4 MCF-7 cell proliferation assay 59 2.5 Measurement of concentrations of estrone, estradiol, icaritin and desmethylicartin and estrogenic activity in serum after oral ingestion of a traditional Epimedium pubescens decoction or estradiol valerate by human subjects 60 2.5.1 Subjects 60 2.5.2 Preparation of the Epimedium pubescens decoction 60 2.5.3 Methodology 62 2.5.4 Blood sampling 63 47 2.5.5 Liquid chromatography tandem mass spectrometry measurement of estrone and estradiol in serum after oral ingestion of a traditional Epimedium pubescens decoction or estradiol valerate by human subjects 63 2.5.6 Statistical analysis for clinical trial 64 2.5.7 Measurement of icaritin and desmethylicaritin in serum after oral ingestion of a traditional Epimedium pubescens decoction by human subjects using gas chromatography–mass spectrometry 65 2.5.7.1 Extraction and preparation of serum samples 65 2.5.7.2 Instrumentation 2.5.7.3 Validation of GC-MS method 2.6 66 67 Development and validation of a liquid chromatography–tandem mass spectrometry assay for simultaneous measurement of five Epimedium prenylflavonoids in rat sera 2.6.1 Animal study protocol 68 68 2.6.2 Sample preparation 2.6.3 Instrumentation 69 2.6.4 Assay validation of LC-MS/MS method 71 2.6.5 Measurement of prenylflavonoids in rat sera 73 2.6.6 Measurement of estrogenicity of rat sera 2.7 69 75 Global gene expression profiling 77 2.7.1 Cell Culture 77 2.7.2 RNA extraction 77 2.7.3 cRNA synthesis and Illumina whole genome chip hybridization 78 2.7.4 Statistical analysis 79 2.7.5 Connectivity Map analysis 80 2.7.6 Real-time PCR 81 48 2.1 Reagents Chemicals Description • • • • • • • • • • • • • • • • • • • • DL-aminoglutethimide apigenin coumestrol dimethyl sulfoxide estradiol estriol estrone genistein hydroxycortisone 4-hydroxytamoxifen kaempferol luteolin progesterone pyridine quercetin raloxifene 4-methylumbelliferone 4-methylumbelliferyl sulphate 4-methylumbelliferyl glucuronide testosterone • • • diarylpropionitrile ICI 182,780 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5triyl)trisphenol • • • • • desmethylicaritin icariin icariside I icarside II icaritin • estradiol valerate Source Sigma (St Louis, MO, USA) Tocris Bioscience (Ellisville, MO, USA) Dr Willmar Schwabe Pharmaceuticals (Karlsruhe, Germany) Schering GmbH (Berlin, Germany) Solvents • • • • acetonitrile ethanol methanol ethyl acetate Merck (Darmstadt, Germany) 49 Description Source Purified helium gas (purity 99.9999%) Soxal (Singapore) Gas • Derivatizing agents • bis-(trimethylsilyl) trifluoroacetamide • N-methyl-N-(trimethylsilyl) trifluoroacetamide Supelco (Bellefonte, PA, USA) Pierce (Rockford, IL, USA) Cell culture reagents • • • • • • Eagle’s minimal essential medium pooled rat serum pooled male human serum sodium bicarbonate sodium pyruvate trypsin • • • • • fetal bovine serum G418 hygromycin B insulin L-glutamine Sigma (St Louis, MO, USA) Gibco Invitrogen (Carlsbad, CA, USA) RT-PCR probes • • GREB1 (Hs00536409_m1) human 18S ribosomal RNA (4319413E) Applied Biosystems (Carlsbad, CA, USA) Enzymes • • • β-glucoronidase/sulphatase from Helix pomatia, type H5 Sulphatase from Acetobacter aerogenes β-Glucoronidase from Escherichia coli Sigma (St Louis, MO, USA) Cells • • HeLa cervical cancer cells MCF-7 breast cancer cells American Type Culture Collection (Manassas, VA, USA) 50 Description Equipment • Glomax 96-well microplate luminometer • API triple-quadrupole LC-MS/MS system • Agilent 6890N GC coupled to a 5975 inert XL mass selective detector Agilent 1200 LC system coupled to an API3200 triple-quadrupole mass spectrometer with Turbo V source and TurboIonSpray and APCI probes 150mm × 2.0 mm (i.d.) Phenomenex Synergi 4µMax-RP column HP-5MS capillary column (5% phenyl methylsiloxane) (30 m × 0.25 mm × 0.25 àM film thickness) ã ã ã Source Promega (Madison, WI, USA) ABI-Sciex (Foster City, CA, USA) Agilent Technologies (Santa Clara, CA, USA) Phenomenex (Torrance, CA, USA) J&W (Santa Clara, CA, USA) • ABI Prism 7000 Sequence Detection System Applied Biosystems (Carlsbad, CA, USA) • Eppendorf microcentrifuge 5424 Eppendorf (Hamburg, Germany) • ND-1000 Spectrophotometer NanoDrop (Waltham, MA, USA) • Illumina BeadArray Reader (Illumina) Illumina (San Diego, CA, USA) 51 Software Description Source GraphPad Software (La Jolla, CA, USA) • GraphPad Prism version 4.0 • Microsoft Excel software Microsoft (Redmond, WA, USA) • Stata version software Stata Corp (College Station, TX, USA) • Illumina BeadStudio software • Genesifter software • Analyst 1.4.2 software Illumina (San Diego, CA, USA) Geospiza (Seattle, WA, USA) ABI-Sciex (Foster City, CA, USA) Assays & RNA/DNA Extraction Kits • Luciferase assay system Promega (Madison, WI, USA) • CyQUANT cell proliferation assay kit Sigma Invitrogen (Carlsbad, CA, USA) • Illumina Sentrix HumanRef-8 V2 BeadChip Arrays • RNeasy Mini Kit • Illumina TotalPrep RNA Amplification Kit Ambion (Carlsbad, CA, USA) • Superscript III Reverse Transcriptase Gibco Invitrogen (Carlsbad, CA, USA) • TaqMan Universal PCR MasterMix Applied Biosystems (Carlsbad, CA, USA) Illumina (San Diego, CA, USA) QIAGEN (Hilden, Germany) 52 2.2 Cell lines and cell culture HeLa cervical cancer cells and MCF-7 breast cancer cells were purchased and cultured as recommended by the American Type Culture Collection (ATCC) Cells were maintained in phenol red-free Eagle’s Minimal Essential Medium supplemented with sodium pyruvate, mM L-glutamine, sodium bicarbonate, and 5% dextran-coated, charcoal-treated fetal bovine serum in humidified atmosphere of 5% carbon dioxide in air at 37°C ERα and ERβ stable cells (described in Section 2.3 below) were maintained in same cell culture media that contains 100 µg/mL hygromycin B and 300 µg/mL G418 MCF-7 breast adenocarcinoma cells were maintained and grown as monolayer cultures in phenol red-free Eagle’s Minimal Essential Medium supplemented with 5% complete fetal bovine serum and 10 μg/mL insulin in a humidified atmosphere of 5% carbon dioxide in air at 37°C 53 2.3 ERα and ERβ cell-based bioassays 2.3.1 Establishment of ERα and ERβ stable cells (from Yap et al., 2005) Determination of LD90 of antibiotics in HeLa cells To successfully generate stable cell lines expressing ER and ERE4 genes, the minimum concentration of hygromycin B and G418 required to kill the untransfected HeLa cells were determined HeLa cells plated in 96-well plate were allowed to adhere overnight Next, the cells were exposed to various doses of hygromycin B and G418 (0, 50, 100, 200, 300, 400 and 500 µg/mL) The selective media was replenished every days and the viable cell counts were determined after 10 days of incubation The lethal dose of the antibiotics to kill 90% of untransfected cells (LD90) was determined ERE4 stable transfection HeLa cells were seeded in a 6-well tissue culture plate and stably transfected with ERE4 plasmid using lipofectamine Sixteen hours post-transfection, the cells were washed, trypsinized and split at ratio of in 10, in 100 and in 1000 into selective media containing 100 μg/mL of hygromycin B The diluted cells were re-seeded onto a new 6well plate and allowed to grow in selective cell culture media for to weeks The selective media were refreshed every days, until hygromycin-resistant foci can be identified The selected clones were screened by transiently transfecting ERα or ERβ plasmid and exposed the cells to estradiol at 10 nM The positive clones were frozen in liquid nitrogen tank for future use 54 ERα/β stable transfection The positive ERE4 clones were seeded in a 6-well plate and stably transfected with pGFP-N2-ERα or pGFP-N2-ERβ plasmids using lipofectamine Sixteen hours posttransfection, the cells were washed, trypsinized and split at ratio of in 10, in 100 and in 1000 into selective cell culture media containing 100 μg/mL of hygromycin B and 300 μg/mlL of G418 The diluted cells were re-seeded onto a new 6-well plate and allowed to grow in selective cell culture media for to weeks The selective media were refreshed every days, until hygromycin/neomycin-resistant foci can be identified The selected clones were screened by exposing the cells to estradiol at 10 nM The positive clones were optimized to achieve maximum fold induction and the good clones were frozen in liquid nitrogen tank for future use 2.3.2 Optimization of ERα and ERβ cell-based bioassay parameters ERα and ERβ cells were developed by Yap et al., 2005 and optimization of bioassay parameters was performed by seeding cells at various densities (0.25, 0.5, 1, and × 104 cells per well) in 96-well plates and incubated for various time intervals (18, 24, 36, and 48 h) to select assay conditions that will give the highest fold increase in estrogen-induced luciferase activity relative to vehicle The optimal cell plating density for ERα and ERβ cells was determined to be × 104 cells and 0.5 × 104 cells per well, respectively The optimal incubation period was 30 h for ERα or 24 h for ERβ cells 55 2.3.3 Validation of stable ERα- and ERβ-driven reporter gene bioassays Inter- and intra-assay coefficients of variance (CV) values were determined using 0.1 or 0.5 nM estradiol Intra-assay CV is the relative SD of the mean of eight determinations in a single assay Inter-assay CV is the relative SD from the mean of three determinations done on 10 different occasions The detection limit is the concentration of the ligand inducing luciferase activity equivalent to the mean plus SD of the vehicle value (Legler et al., 1999) 56 2.5.7.3 Validation of GC-MS method Linearity of calibration curves was studied by using control pooled male human serum (Sigma) which were analyzed in full scan and SIM mode to verify the absence of genistein, icaritin and desmethylicaritin This control pooled male human serum was spiked with increasing doses of icaritin and desmethylicaritin to give final concentrations ranging from 0.15 to 10 nM All standards contained 10 nM of genistein Calibration standards were tested and found to be stable at −20oC under nitrogen gas for at least months A calibration curve with at least six concentration points was constructed every experimental day The least regression method and the squared correlation coefficient (R2) were used to estimate linearity LOD and LOQ are defined as: LOD= 3.3σ/S, LOQ=10σ/S, where σ = SD of the background response and S = the slope of the calibration curve (E.M Agency, 2005) The background response was measured by analyzing control pooled human sera and SD was calculated from 10 independent assays The average of the slope of four independent calibration curves was used as S for LOD and LOQ calculation To ascertain accuracy and imprecision of the method, low, medium and high QC samples, containing 0.15, 0.5 or nM of icaritin and desmethylicaritin, respectively and internal standard in pooled human sera, were placed at each assay for determination of accuracy and imprecision (Shah et al., 2000) Accuracy and imprecision were obtained from six independent assays within one experimental day (intra-day) or independent assays from five consecutive days (inter-day) 67 2.6 Development and validation of a liquid chromatography–tandem mass spectrometry assay for simultaneous measurement of five Epimedium prenylflavonoids in rat sera 2.6.1 Animal study protocol Female Sprague-Dawley rats, ovariectomized at weeks of age, were treated with 300 mg of Epimedium standardized preparation per kilogram of body weight at weeks of age by gavage The Epimedium preparation (PSC1929/L01-60/B/Wo 06-002-28) was derived from Epimedium brevicornu dry leaves that were extracted with 60% ethanol The preparation contained 143.3 mg/g of icariin, 0.157 mg/g of icariside I, 18.68 mg/g of icariside II, 0.009 mg/g of icaritin, and 0.122 mg/g of desmethylicaritin, respectively Following Epimedium administration, blood sampling was performed at 0, 0.5, 1, 2, 4, 8, 24, 48, and 72 h time points (four rats/each time point) Sera were separated and stored at −80oC until analysis Animal experiments were performed in the laboratories of Dr Willmar Schwabe Pharmaceuticals in accordance with German Federal law for animal welfare Written approvals for the study were granted by veterinary authority of BadenWurttemberg, Germany; and the Institutional Animal Care and Use Committee, National University of Singapore Rats were fed a soy-free diet 68 2.6.2 Sample preparation All serum samples were thawed at room temperature, spiked with internal standard (coumestrol, 100 nM) and equilibrated at 37◦C for h before extraction Analytes were extracted together with internal standard from 0.5 mL serum aliquots thrice via liquid–liquid partition (1 mL × 3) using water-saturated ethyl acetate for inside a mL polypropylene centrifuge tube After each round of solvent–solvent partition, the mixture was centrifuged for at 10,000 g using the Eppendorf microcentrifuge 5424 (Eppendorf) to clearly separate the organic layer from the aqueous layer The organic layers obtained from the three rounds of solvent–solvent partition were combined and dried with a gentle stream of nitrogen gas The residue was solubilized using 100 µL of methanol by vortexing for This was then transferred into a 200 µL borosilicate glass insert placed inside a sample vial for LC–MS/MS analysis 2.6.3 Instrumentation Epimedium brevicornu extract contains compounds such as icariin, icariside I, icarside II, icaritin and desmethylicaritin and metabolism of these compounds, particularly, the glycosides, occurs upon the ingestion of the extract To understand the bioavailability and conversion of these compounds after ingestion, a LC–MS/MS analytical method was hence developed and validated to measure the levels of icariin, icariside I, icariside II, icaritin and desmethylicaritin using Agilent 1200 LC system (Palo Alto, CA, USA) coupled to an API3200 Triple-Quadrupole Mass spectrometer (Applied Biosystems/MDS SCIEX) A Turbo V source with TurboIonSpray and APCI probes were used for the analysis Data acquisition was performed with Analyst 1.4.2 software (AB MDS Sciex) 69 The chromatographic separation was performed on 150mm ì 2.0 mm (i.d.) Phenomenex Synergi 4àMax-RP column (Phenomenex) with a column oven temperature of 35oC The mobile phase consisted of two eluents, namely, solvent A (ultrapure water containing 0.5 mM ammonium formate, pH 3) and solvent B (acetonitrile flow rate of 0.4 mL/min Before every run, the column was first equilibrated with 45% of solvent B for before sample injection The gradient was programmed to increase from 45% to 90% of solvent B in and maintained at 90% of B for to complete the separation Standards or samples were introduced into the LC using an Agilent 1200 G1367B autosampler and injection volume was 10.0 µL The ion source was operated in the negative mode The experiment was divided into two periods to detect two groups of analytes: prenylflavonoid glycosides (0.2–4.5 min) and aglycones (4.51–8 min) Ion-spray voltage and temperature were set constantly at –4500 V and 550oC for both periods 70 2.6.4 Assay validation of LC-MS/MS method Linearity was studied by first analyzing commercial pooled rat serum using full scan and MRM mode to confirm the absence of interference with the internal standard and the five analytes To generate calibration samples, increasing doses of icariin, icariside I, icariside II, icaritin and desmethylicaritin were added to 0.5 mL of pooled rat sera to obtain final concentrations ranging from 0.78 to 100 nM All standards contained 100 nM of coumestrol as the internal standard Six calibration curves with seven concentration points were constructed The final curves for each of the five compounds were generated with the average values and were used for quantification of the analytes The least squares regression method was used to estimate linearity LOD and LOQ were first determined by using the statistical method based on the calibration curve constructed from standard compounds The LOD and LOQ were defined as LOD = 3.3σ/S,LOQ=10σ/S, where σ is SD of the background response and S is the slope of the calibration curve (E.M Agency, 2005) The background response was measured by analyzing pooled rat sera and SD was calculated from 10 independent assays The average of the slope of six independent calibration curves was used as S for LOD and LOQ calculations The empirical method for estimation of the LOD and LOQ involved analyzing decreasing concentrations of analytes The LOD was defined as having a S/N of 3:1, and a S/N of 10:1 was defined for LOQ (U.S Food and Drug Administration, 1996) Three independent assays were performed to obtain the S/N Accuracy and imprecision of the method were studied by using QC samples consisting of a high, medium and low concentration of the five analytes, containing 1, and 10 nM of icariin, icaritin and desmethylicaritin; 5, 30 and 60 nM of icariside I and 71 icariside II, respectively and internal standard in pooled rat sera, were placed at the beginning and end of each assay for determination of accuracy and imprecision (Shah et al., 2000) Accuracy and imprecision of the method were obtained from five independent assays within one experimental day (intra-day) or independent assays from consecutive days (inter-day) Matrix effect was studied using 0.5 mL of blank rat serum that was extracted three times with mL of ethyl acetate The ethyl acetate layers were combined and dried with nitrogen gas The residue was reconstituted with 0.5 mL of methanol to serve as serum extract Mixture of five analyte standards and internal standard were spiked into 0.1 mL of methanol (neat solution) and 0.5 mL of serum extract, respectively Neat solution samples were directly injected and analyzed with LC–MS/MS Samples spiked in serum extract were dried with nitrogen gas and reconstituted with 0.1 mL of methanol and analyzed with LC–MS/MS Matrix effect was assessed by comparing the peak areas of the analytes and internal standard between neat solution and serum extract (Matuszewski et al., 2003) Each assay was performed with five replicates Stability was examined by using analyte standards that were spiked into blank serum with low and high concentrations and stored at −80oC for month The samples were thawed, processed and analyzed with the developed method The concentrations calculated from the calibration curve were compared with actual values to determine the long-term stability (Sarkar et al., 2008) For post-preparative stability study, sera spiked with low and high levels of standards were processed and kept at 4oC in an autosampler for 24 h before analysis (Sarkar et al., 2008) Concentrations of the samples were compared with actual values All the assays were performed in five replicates 72 2.6.5 Measurement of prenylflavonoids in rat sera Flavonoids and internal standard in sera were analyzed with full scan mode to generate the precursor deprotonated molecules [M−H]− and the most abundant and characteristic product ions were selected for MRM analysis The ion pairs selected for the five flavonoids and internal standard were 513→351 for icariin, 529→367 for icariside I, 513→351 for icariside II, 367→175 for icaritin, 353→136 for desmethylicaritin, and 267 →211 for internal standard, respectively Coumestrol was chosen as the internal standard, because it has similar chemical and physical properties to Epimedium flavonoids, but is not naturally present in Epimedium and the rat diet The LOD and LOQ for icariin, icariside I, icariside II, icaritin and desmethylicaritin were < nM and 1–2 nM respectively Inter- and intra-assay variabilities were < 15% and accuracies were between 94% and 114% respectively The linearity of the calibration curves was good (R2 > 0.99) within the concentration range of 0.78–12.5 nM for icariin, icaritin and desmethylicaritin, and 0.78–100 nM for icariside I and II To determine the concentration of conjugated flavonoids, sera were exposed to enzyme mixture from Helix pomatia, type H5 (Sigma) before LC–MS/MS analysis Freshly prepared enzyme solution in water with a pH ~7 (equivalent to ~2000 U of glucuronidase and ~200 U of sulphatase) was added to 0.5 mL of serum No buffer was used for enzymatic digestion The H pomatia enzyme mixture was dissolved in ultrapure water which was then vortexed to mix and finally added to serum to start the enzymatic hydrolysis process The mixture was vortexed briefly and incubated at 37oC for h To determine completeness of enzymatic digestion, µM of 4-methylumbelliferyl sulfate and 4-methylumbelliferyl glucuronide were spiked into commercial rat serum, exposed to enzyme and concentrations of 4-methylumbelliferone and its conjugates were measured 73 by LC–MS/MS Enzyme hydrolysis was performed in water without buffering reagent and acidifying the rat serum because prenylflavonoids precipitated under acidic conditions (pH 5) The enzymatic digestion efficiencies of the H pomatia enzyme mixture for both 4-methylumbelliferyl sulfate and 4-methylumbelliferyl glucuronidewere 100% after incubation at 37oC for h The enzyme hydrolysis was terminated by liquid– liquid partition and prenylflavonoid aglycones were found to be stable under the postpreparative conditions (Shen et al., 2009) 74 2.6.6 Measurement of estrogenicity of rat sera Estrogenicity of flavonoids in mixtures was measured using HeLa cells stably expressing ERα and ERβ proteins Pooled rat sera used for the construction of the calibration curve were thawed at room temperature For measurement of bioactivity, samples were thawed at room temperature and immediately reconstituted with Eagle’s Minimum Essential Medium to a final concentration of 20% sera These reconstituted samples were then exposed to ERα and ERβ stable cells without further treatment To measure the bioactivity conferred by conjugated flavonoids and other constituents, serum samples were first hydrolyzed by adding µL of β-glucuronidase from Escherichia coli (Sigma) containing 25,000 U of βglucuronidase and µL of sulphatase from Aerobacter aerogenes (Sigma) containing 0.020 U sulphatase into 100 µL of rat serum sample The mixture was then incubated for h at 37oC This enzyme mixture was used instead of glucuronidase from H pomatia (type H5) because the latter has been reported to contain contaminating amounts of estrogenic isoflavones (Taylor et al., 2005) Isoflavones from H pomatia H5 preparation did not interfere with the measurements of prenylflavone derivatives from Epimedium To ensure complete enzymatic digestion using this method, the hydrolysis of 4methylumbelliferyl sulfate and 4-methylumbelliferyl glucuronide in rat sera was monitored using LC–MS/MS Result of monitoring showed that by h at 37oC, the two conjugates were completely hydrolyzed and the two conjugates were not detected The area under the concentration–time and bioactivity–time curves (AUC) of flavonoids was calculated by the trapezoidal method (Atkinson Jr et al., 2006) Only the 0–24 h period was used for AUC calculations in order to reduce variability due to long sampling intervals after this time period The maximum plasma concentration (Cmax), 75 maximum estrogenic effect (Emax) and the time to reach these maxima (tmax) were obtained by visual inspection of the experimental data Estrogenic activity in serawas reported as estradiol equivalents (pM E2) based on calibration curves constructed with spiked estradiol Values below the detection limit of the bioassay were scaled to Comparison of estrogenic effects between treatments was performed by calculating AUC and these were reported as pM E2 h−1 76 2.7 Global gene expression profiling 2.7.1 Cell Culture MCF-7 cells were first grown in phenol red-free Eagle’s Minimal Essential Medium cell culture medium containing 10% charcoal-stripped fetal bovine serum in a monolayer culture for a week, with medium change performed on every alternate day After the days, the cells were trypsinized, seeded into 12-well plates at a density of x 105 cells per well and allowed to adhere overnight Cell culture medium was then decanted and cells were incubated with test compounds dissolved in phenol red-free cell culture medium containing 5% dextran-coated, charcoal-stripped fetal bovine serum for h Each treatment was performed in triplicates 2.7.2 RNA extraction After a h treatment period, cells were rinsed with phosphate saline buffer, harvested and total RNA was extracted using the RNeasy Mini Kit (QIAGEN) as described by the manufacturer, including the QIAshredder spin column procedure and oncolumn RNase-free DNase digestion RNA was eluted with 40 μL of RNA-storage solution RNA QC and quantification were determined using the ND-1000 Spectrophotometer (NanoDrop) Only RNA with a quality ratio A260/A280 between 1.9 and 2.1 and no evidence of peak degradation (18s/28s) was used RNA samples were then aliquoted and stored at −80 °C until use 77 2.7.3 cRNA synthesis and Illumina whole genome chip hybridization Gene expression analysis was executed using Illumina Sentrix HumanRef-8 V2 BeadChip Arrays (Illumina) allowing the analysis of ~23,000 transcripts First, total RNA was amplified to yield biotinylated cDNA and then purified using the Illumina TotalPrep RNA amplification kit (Ambion) according to the manufacturer's instructions Briefly, 550 ng of total RNA was reverse-transcribed to cDNA using a T7 oligo(dT) primer Second strand cDNA was synthesized, in-vitro transcribed and labeled with biotin-NTP After purification, the cDNA was quantified using the ND-1000 spectrophotometer Seven hundred and fifty nanograms of amplified biotinylated cRNA were hybridized onto the Illumina Sentrix BeadChip in a hybridization cartridge under humidified conditions for 20 h at 58°C in a hybridization oven The chips were then washed, stained for 10 with μg/mL streptavidin-conjugated Cy3, and finally dried by centrifugation according to the protocol provided Fluorescence detection was carried out by confocal laser scanning with the Illumina BeadArray Reader (Illumina) at 532 nm and 0.8 μm resolution 78 2.7.4 Statistical analysis The array data analysis is a multi-step process beginning with decoding the image spots because of random assembly of the microbeads on the array surface (Gunderson et al., 2004; Kuhn et al., 2004) Each bead type is represented on average 30 times per array providing internal replicates (Steemers & Gunderson, 2005) Illumina BeadStudio Software was used for condensing these data and further to ensure array quality based on different control bead parameters Data were extracted and normalized using the rank invariant method available in the BeadStudio software (Illumina) Genesifter software (Geospiza) was used to identify differential gene expression due to ER-dependent regulation, microarray data from triplicate experiments were subjected to ANOVA by using the criteria where a regulated gene is defined to be one that was activated by at least 2-fold or greater and statistically different from the control cells treated with vehicle where the adjusted p-value using the Benjamini and Hochberg procedure (BH-adjusted p-value) is less than 0.0001 (Benjamini & Hochberg, 1995) 79 2.7.5 Connectivity Map analysis Connectivity Map (build 02) contains 6100 gene expression profiles of cell lines treated with 1309 distinct small molecules (Lamb et al., 2006) Data from Connectivity Map was downloaded from the Connectivity Map main website (http://www.broadinstitute.org/cmap/) Functional connections between the query gene signatures and database gene signatures induced by small molecules were explored using the Connectivity Map database The upregulated genes were grouped and their probe sets were consolidated to give the ‘up’ tag file and likewise was done for downregulated genes These two files were used to query the Connectivity Map database, and the results showed the most significant similarities and dissimilarities to the database profiles Data were evaluated using the Kolmogorov-Smirnov statistic, a nonparametric, rank-based pattern-matching strategy Each reference signature in the database was scored according to its similarity with the query signature, and the extent of the similarity is described as the “connectivity score.” The connectivity score ranges from +1 to −1; nearer to +1 means higher similarity, means no similarity, and nearer to −1 means opposite similarity For the query, the probe ID defined by the Affymetrix GeneChip Human Genome U133A array was used 80 2.7.6 Real-time PCR Total RNA from treated MCF-7 cells were isolated using QIAGEN’s RNAeasy kit Reserve transcription of RNA was then carried out with Superscript III Reverse Transcriptase (Gibco Invitrogen) according to the manufacturer’s protocol with random primers Quantitative PCR amplification and detection were performed with TaqMan Universal PCR MasterMix according to the manufacturer’s protocol on ABI Prism 7000 Sequence Detection System (Applied Biosystems) The gene expression assay kit for GREB1 (Hs00536409_m1) and human 18S ribosomal RNA (rRNA) as an internal control were used Results are presented as the fold increase compared to the vehicle Values are the mean ± SE of two independent experiments 81 ... analytes, containing 1, and 10 nM of icariin, icaritin and desmethylicaritin; 5, 30 and 60 nM of icariside I and 71 icariside II, respectively and internal standard in pooled rat sera, were placed... using lipofectamine Sixteen hours posttransfection, the cells were washed, trypsinized and split at ratio of in 10, in 100 and in 1000 into selective cell culture media containing 100 μg/mL of hygromycin... fetal bovine serum and 10 μg/mL insulin in a humidified atmosphere of 5% carbon dioxide in air at 37°C 53 2.3 ERα and ERβ cell- based bioassays 2.3.1 Establishment of ERα and ERβ stable cells (from