Transcription profiling in breast cancer cell lines after Ctet treatment

ABSTRACT: Cyclic indole-3-carbinol (I3C) tetrameric derivative (CTet) is an anticancer molecule that has been shown to exert an antiproliferative activity in both MCF-7 and MDA-MB-231 breast cancer cell lines. To characterize the molecular mechanisms leading to the inhibition of the cell proliferation, gene expression analyses were conducted. MCF-7 and MDA-MB-231 breast cancer cells were plated in 6-wells culture plates at density of 150,000 cells/well and cultured overnight. Cellular treatments were conducted at 6uM and 12 uM concentration of CTet, or vehicle control, for 24 h. Cell survival was evaluated by trypan blue dye exclusion assay and, after washing in phosphate buffered saline (PBS), the cells were pelletted by centrifugation and stored at -20°C with 300µl of RNA-later solution (Sigma-aldrich). Total RNA was purified from treated and control cells using the RNeasy plus kit (Qiagen). Biotin-labeled cRNA was synthesized using the CodeLink iExpress Assay reagent kit (GE Healthcare), following the manufacturer’s protocols. Biotin-labeled cRNA obtained from each biological sample was fragmented and hybridized against three independent arrays (10 ug each) at 37°C for 22 h. After hybridization, the arrays were washed, stained with Cy5-streptavidin and scanned using a ScanArray GX scanner (Perkin Elmer), with a resolution of 5 um. The image files generated by the scanner were processed using the Codelink Expression Analysis software (GE Healthcare). Normalized data from the Codelink software package were analyzed with GeneSifter software (www.genesifter.net; Geospiza Inc., Seattle, WA) for statistical validation and data mining. Normalized data of the two experiments were subjected to analysis of variance (ANOVA) and 5% false discovery rate calculation (Benjamini and Hochberg). The cut-off parameters for differential gene expression were p =0.01 and fold change threshold =2.

ORGANISM(S): Homo sapiens

SUBMITTER: Galluzzi L 

PROVIDER: S-ECPF-MEXP-2989 | biostudies-other | 2012

REPOSITORIES: biostudies-other