Moreover, in estrogen deprived medium, tamoxifen can act as an agonist towards ER, adding another complicating factor to the mechanis tic interpretation of tamoxifen resistance. We used a phe nol red free DMEM medium containing 5% FBS so AZD9291 astrazeneca that the background estrogen level is in a range that is unli kely to induce adaptive changes due to estrogen depriva tion and to minimize the agonistic action of tamoxifen in ER breast cancer cells. In this study, we examined global proteomic altera tions of the tamoxifen resistant cell line vs the parental MCF 7 cells using an isobaric labeling approach com bined with a high resolution tandem mass spectrometry instrument for relative quantitative analysis. Our proteo mics data demonstrated extensive adaptive changes in the proteome involving hundreds of significantly up and down regulated proteins.
In particular, results from this study revealed the overexpression of multiple tumorigenic, pro metastatic proteins and the down reg ulation of ER mediated signaling pathways. These find ings Inhibitors,Modulators,Libraries provide novel insights into the complex events of the adaptive signaling network occurring during the acquisition of tamoxifen resistance in breast cancer cells and highlight the role of S100P in conferring both resis tance and enhanced Inhibitors,Modulators,Libraries migration. Materials and methods Cell culture MCF 7 cell line was purchased from ATCC, and routinely cultured in phenol red free DMEM medium supplemented with 5% FBS, 4 mM glutamine, 1 mM sodium pyruvate, 100 IU mL penicillin, 100 ug mL streptomycin and 0. 25 ug mL amphotericin.
Tamoxifen resistant Inhibitors,Modulators,Libraries variant cells derived from MCF 7 cells were continuously cul tured in the medium as described above containing addi tional 10 7 M 4 OH Tam Inhibitors,Modulators,Libraries for at least six months, along with the parental MCF 7 cells under identical culture conditions except that the control cells were treated with 0. 1% ethanol. The two cell lines were grown side by side at all times. Cul tures were maintained in 5% carbon dioxide at a tem perature of 37 C. Cell growth assay For growth assay in the presence of 10 Inhibitors,Modulators,Libraries 7 M 4 OH Tam, MCF 7 cells cultured with 10 7 M 4 OH Tam for zero to six months were plated in six well plates at a density of 50,000 in each well in 5% FBS DMEM medium. The cells were then treated with 10 7 M sellectchem 4 OH Tam for five days, while equal treatment volumes of ethanol were used as a vehicle control. Cell numbers were counted with a Coul ter instrument. The ratio of 4 OH Tam treated cell numbers to vehicle treated cell numbers was defined as survival ratio. Experiments were conducted in triplicate and data repre sented as mean SD.