HER2 and progesterone receptor at recurrent and initial examination was obtained from individual pathological studies. Antibodies Tipifarnib R115777 for detecting p110a, p110b, p110g, phosphatase and tensin homolog, Akt1, Akt2, Akt3, phospho Ser473 Akt, mTOR, S6 protein kinase 1, phospho Thr 389 S6 protein kinase 1, S6, phospho Ser235/236 S6, p44/42 mitogen activated protein kinase and phospho Thr202/ Tyr204 p44/42 MAPK were from Cell Signaling Technology. Cell growth analysis and calculation of 50% inhibitory/lethal concentrations To determine the effects of estradiol and fulvestrant on the growth of LTED cells, the cells growing in CSS medium were plated in 96 well Optilux dishes and were treated without or with fulvestrant or the indicated concentrations of 17b estradiol on the afternoon after plating. The medium was replenished every three to four days and cell development was assessed after 7 days by measuring Alamar Blue reduction with a fluorescent microplate reader. For calculation of the half maximal inhibitory concentration and the 500-point life-threatening concentration, cells were cultured in phenol red free RPM1 1640 containing five hundred CSS for at least a week just before plating in 96 well Optilux meals for drug treatment. Cellular differentiation Alternately, cells developing in phenol red RPMI 1640 medium containing 10 % FBS were then switched to CSS medium and plated in 96 well Optilux dishes for at least 1 week before drug treatment. Five dilutions of each drug were made employing a 1,5 serial dilution. Solutions were performed in triplicate and the studies in each cell line were performed at least twice. The effect of solutions on cell viability were assessed 0 hours and 96 hours after drug exposure by measuring the Alamar Blue reduction using a fluorescent microplate reader. Cell growth was analyzed using GraphPad Prism version 5. 00 for Windows. The fitted curves were then used to find out the IC50 and LC50 values. Apoptosis analysis To evaluate apoptosis, cells growing in CSS medium were treated as indicated for 4 buy CX-4945 days. For treatments using fulvestrant, cells were pre-treated with fulvestrant for 3 days prior to therapy with estradiol or PI3K inhibitors to make certain sufficient down-regulation of the ER. Floating and adherent cells were then collected and marked to identify apoptotic cells using the APO BrdU TUNEL Assay Kit in accordance with the manufacturers instructions. For each sample, no less than 10,000 activities were acquired over a Cytomics FC500 move cytometer and data were analyzed using FlowJo software. Individual samples Human cyst samples from patients with recurrent or metastatic breast cancer were obtained under the auspices of an Institutional Review Board approved protocol in the Siteman Cancer Center. Informed consent was obtained from all patients involved.