Introduction Heat shock protein 90 is a highly conserved chaperone protein

Materials and methods Materials Acetyl CoA, Malonyl CoA, NADPH, DMSO, and mangostin were purchased from Sigma. Dulbeccos modified Eagles medium and fetal bovine serum were purchased from Life Tech nologies, Inc. FAS anti body was obtained from BD Pharmingen. FAK, phosphor FAKtyr397, AKT, phospho AKTSer473, ERK12, phosphor buy JNJ-7706621 ERK12Thr202Tyr204, Bax, Bcl 2, PARP and GAPDH were purchased from Cell Signaling Technol ogy. Cell lines and culture The human breast epithelial cell lines MCF 7, estrogen receptor positive cells derived from an in situ carcinoma, and MDA MB 231, estrogen receptor negative cells de rived from a metastatic carcinoma, were used in the study. The cells were purchased from the American Type Culture Collection and were grown in DMEM supplemented with 10% fetal bovine serum.<br><br> Cells were maintained at 37 C in a hu midified atmosphere of 95% air and 5% CO2. Cell viability assay Cell viability was assessed purchase LDN193189 by Cell Counting Kit assay as previ ously described. Briefly, cell were seeded at a con centration of 1 104 cells200 ulwell into 96 well plates, and allowed an overnight period for attachment. Medium was removed and fresh medium along with various concentrations of mangostin were added to cultures in parallel. Following treatment, drug free medium and 10 ul CCK 8 solution were added and cells were incubated for 1 h at 37 C. The op tical density value was measured at 450 nm by a microplate spectrophotometer. All experiments were performed in quadruple on three separate occasions. Analysis of apoptosis Cell apoptosis detection was performed using an Annexin V FITC Apoptosis Detection Kit according to the manufacturers protocol.<br><br> Briefly, cells were collected after 24 h treatment with mangostin. LY2228820 The cells were washed twice with cold PBS then resuspended in 1binding buffer at a concentration of 1 106 cellsml. Then 500 ul cell suspension was incu bated with 5 ul Annexin V FITC and 10 ul PI for 15 min in the dark and analyzed by a FACScalibur instrument within 1 h. Apoptotic cells were those stained with Annexin V PI plus Annexin V PI. FAS activity assay After 24 h of exposure to mangostin, cells were har vested by treatment with trypsin EDTA solution, pelleted by centrifugation, washed twice, and resuspended in cold phosphate buffered solution. Cells were sonicated at 4 C and centrifuge at 13,000 rpm for 30 min at 4 C to ob tain particle free supernatants.<br><br> FAS activity was deter mined spectrophotometrically at 37 C by measuring the decrease of absorption at 340 nm due to oxidation of NADPH as previously described. 50 ul Particle free supernatant, 25 mM KH2PO4 K2HPO4 buffer, 0. 25 mM EDTA, 0. 25 mM dithiothreitol, 30 uM acetyl CoA, 350 uM NADPH in a total volume of 500 ul were monitored at 340 nm for 100 s to measure background NADPH oxidation. After the addition of 100 uM of malonyl CoA, the reaction was assayed for an additional 1 min to determine FAS dependent oxidation of NADPH. FAS activity was expressed in nmoles NADPH oxidized min−1 mg protein−1. Immunoblot analysis Following treatment of breast cells with mangostin at the corresponding concentration and for the indicated time, cells were harvested using trypsin EDTA, washed twice with PBS, and stored at −80 C.