aTEA 40 uM 12 hour treatment for MDA-MB-435 cells

In an effort to identify novel signaling molecules modulated by α-TEA, microarray analyses were performed. DNA array data obtained from human MDA-MB-435 breast cancer cells treated with 40 µM α-TEA for 12 h revealed over 400 genes that were consistently either up- or down-regulated. Thirty-four genes were selected based on their possible involvement in the known biological activities of α-TEA, and three: phorbol-12-myristate 13-acetate-induced protein (PMAPI) which codes for the protein NOXA, ABL2 which codes for the protein referred to as ARG (Abelson-related gene) and THBS1 which codes for thrombospondin 1, TSP-1 were chosen for further study. Keywords: Anti-cancer drug (aTEA at 40 uM 12 hour) treatment for MDA-MB-435 cells The whole-genome cDNA microarrays were manufactured in the laboratory of Dr. Vishwanath Iyer (U.T. Austin). For the microarray experiments, MDA-MB-435 cells were cultured with 40 mM of a-TEA or ethanol control for 12 h. Messenger RNA was isolated using the FastTrack 2.0 kit (Invitrogen, Carlsbad, CA) then reverse transcribed by SuperScript II reverse transcriptase (Invitrogen) according to the manufacturers’ instructions. cDNA was purified using MinElute columns (Qiagen, Valencia, CA) and cDNA samples from vehicle treated controls were labeled with Cy3 and samples from a-TEA treated cells were labeled with Cy5. Array hybridizations were performed in humidity chambers at 65°C for 16 h. Slides were then washed, dried, and scanned using an Axon GenePix 4000 scanner (Axon Instruments, Union City, CA). Microarray images were quantified using GenePix 4.0 software (Axon). Data were uploaded to the Longhorn Array Database and filtered to pass minimum quality control thresholds before subsequent analysis. For a single comparison between two groups, a log2-transformed treatment/control signal ratio of 1 or –1 was chosen as the criterion for induction or repression, respectively. Data from 5 replica experiments were combined to eliminate variability and reduce misclassification.