2.1. Cell Culture and Media
Human breast cancer cells (MCF-7 and MDA-MB 231) and immortalized normal human breast mammary MCF-10A cells were obtained from Lombardi Comprehensive Cancer Center cell repository and grown in Dulbecco’s modified Eagle medium (DMEM), supplemented with 5% heat-inactivated fetal bovine serum and 25 μg/ml gentamicin (Invitrogen, Carlsbad, CA).
2.2. Chemicals, Reagents and Antibodies
T3s (>95% pure) were from EISAI Corporation (Woodcliff Lake, NJ) and Carotech (Edison, NJ) and dissolved in DMSO. WST-1 reagent used for cytotoxicity assay and protease inhibitor cocktail tablets (Roche Applied Science, Indianapolis, IN); ECL Plus Western blotting detection system (GE Healthcare, Piscataway, NJ); and Coomassie protein assay reagent (BioRad, Hercules, CA).
The following antibodies were obtained commercially: Cleaved PARP, GRP78/Bip, Chop, PERK, Cyclin D-1, p-IRE1α, IRE1α, p-eIF2α, eIF2α, β-Tubulin, and GAPDH antibodies (Cell Signaling Technology Inc, Danvers, MA); ATF3, ATF6 and p-PERK (Santa Cruz Biotechnology, Santa Cruz, CA); β-Actin from Sigma, and ATF4 (Abcam, Cambridge, MA). Secondary antibodies conjugated with horseradish peroxidase included goat anti-mouse IgG, goat anti-rabbit IgG and rabbit anti-goat IgG (Jackson ImmunoResearch, West Grove, PA).
2.3. Cell Viability and Proliferation Assay
Effects of T3s on cell viability and proliferation of breast cancer cells were determined using a cell viability detection kit (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate, WST-1) according to the manufacturer’s instructions (Roche Applied Science, Indianapolis, IN). Briefly, the cells (MCF-10A, MCF-7 and MDA-MB 231) were seeded onto 96-well plates at a density of 3,000cells per well in six replicates, and maintained overnight in 100 μl of 5% fetal bovine serum-containing medium. The following day, cells were treated for 24, 48 and 72 h with different concentrations of α and γ-T3 ranging from 10 to 40 μM made in complete medium using six wells per treatment condition. Control cells were treated with DMSO at a final concentration of 0.1%. At the end of each treatment after 24, 48 and 72 h, 10 μl of the WST-1 reagent was added to each well. Plates were incubated for 2h at 37°C and analyzed at A= 450/600 using a Bio-Rad Model 680 micro plate reader. Cell viability was assessed by trypan blue dye exclusion assay.
2.4. Cell Cycle Analysis
The effects of γ-T3 on cell cycle were determined by flow cytometric analysis of MCF-7 cells treated with γ-T3. The cells were treated with 40 μM of γ-T3 for 24h in triplicates. The floating cells were pooled with the adherent cells, washed and fixed with 70% ethanol followed by FACS analysis. Briefly, the cells were centrifuged and suspended in 1ml of phosphate-buffered saline containing 50μg/ml each of RNase A and propidium iodide (both from Sigma-Aldrich, St. Louis, MO). The stained cells were analyzed using the FACsort (Becton Dickinson, Franklin Lakes, NJ), and Reproman computer software. The percentage of cells containing sub-G1
DNA content was used as an index of apoptosis as described previously [21
]. Gating was set to exclude cell debris, doublets, and clumps when determining cells in different stages.
2.5. RNA isolation and Gene Expression Microarray
Gene expression pattern in MCF-7 cells treated with 40 μM γ-T3 were analyzed by microarray studies. Four independent experiments were performed with cells grown on different days and from different stocks. The RNA from cells treated with DMSO control and γ-T3 treated cells was isolated using Trizol (Invitrogen) and further purified using Qiagen RNAeasy kit. The RNA concentrations were determined spectrophotometrically using a Beckman DU640 Spectrophotometer (Beckman Coulter, Brea, CA). RNA quality was assessed using the Agilent 2100 Bioanalyzer. High quality RNA was labeled and hybridized to U133A2 Affymetrix GeneChips following manufacturer’s recommendations (Affymetrix, Santa Clara, CA). Microarray data quality was assessed using various tools including those recommended by Affymetrix. All array data presented here passed the quality control measures. Our data analysis began with preprocessing of the probe-level Affymetrix data (.cel files). We used RMA for background adjustment, quantile method for normalization, and a robust multi-array average for summarization. These methods have provided better performance than MAS 5.0 and MBEI in detecting known levels of differential expression using spike-in Affymetrix data [22
]. They are implemented in several statistical tools including Bioconductor and BRB-ArrayTools (NCI, Bethesda, MD).
We analyzed the preprocessed data to identify the genes that were differentially expressed between the untreated and treated groups using random variance model implemented in BRB-ArrayTools. The random-variance t-test is an improvement over the standard separate t-test as it permits sharing information among genes within-class variation without assuming that all genes have the same variance [24
]. The false discovery rate (FDR) was estimated using the method of Benjami and Hochberg [25
]. Probesets were considered statistically significant if their p-value was less than 0.001. With this threshold, 969 probe sets were found statistically significant (p<0.001 and FDR<0.023). These probe sets had a fold change > 1.2. The probe sets with >1.5 fold change were selected for analysis to identify canonical pathways, physiological functions and interaction networks using the Ingenuity Pathway Analysis software (IPA) (Ingenuity Systems, Redwood City, CA).
2.6. Real Time RT-PCR
For Real Time PCR, complementary DNA (cDNA) was synthesized from 1 μg of total RNA using Transcription First Strand cDNA Synthesis Kit (Roche) following the manufacturer’s instructions. The primers were purchased from RealTimePrimers.com (Elkins Park, PA). For each primer set, the meltcurves were performed to verify that the primers amplified a single product and ensure that there were no primer dimers or amplification in the no-template controls. Three independent experiments were performed in triplicate with RNA isolated with different cell stocks, treatments and different days.
PCR amplification was performed with the 7300 Real-Time PCR System (Applied Biosystems, Carlsbad, CA) in 50 μl reactions using 5 μl of cDNA (50 ng of input total RNA), 300 nM each of forward and reverse primer and 1× FastStart SYBR Green PCR Master Mix (Roche). Expression levels of the genes of interest were normalized to GAPDH. The QPCR cycling parameters consisted of 1 cycle of 95°C for 10 minutes, and 40 cycles of (95°C for 15 sec and 60°C for 1 minute). On a given 96- well plate, target and control normalizing genes were run in triplicate. The CT (threshold cycle of amplification) values were determined using the 7300 Real-Time PCR System RQ Study Software (Version 1.3.1) (Applied Biosystems). To determine fold change in expression levels the comparative CT method was used using the formula 2−ΔΔCT.
2.7. Western blotting
Immunoblotting was performed essentially as described previously [26
]. After 24 h treatment with γ-T3, adherent and floating cells were collected. Whole cell extracts (total cell homogenates) were prepared by lysing of cells in radioimmune precipitation assay buffer, and proteins were separated on a 4–20% gradient SDS gel (Pierce), followed by transferring of proteins to polyvinylidene difluoride membranes (0.45 μm, Immobilon-P, Millipore, Billerica, MA). Membranes were immunoblotted with the appropriate primary antibody and peroxidase-conjugated secondary antibody. The antigen-antibody complex was determined using the ECL detection assay (Amersham/GE Life Sciences, Piscataway, NJ). Each Western Blot was repeated at least 3 times. Representative Western Blots are shown.
2.8. Statistical Analyses
Cell proliferation experiments were performed in 6 replicates. Cell cycle and RT-PCR experiments were performed in triplicates. Student’s T test was used to analyze treated vs. untreated cells. Results were expressed as averages ± SD. P<0.05 was considered significant. Statistical analysis of microarray data was performed as described above.