The survival ratio of the tamoxifen treated cell line remained at about 90% from month 3 and beyond. The acquired resistance to tamoxifen was further mea sured by dose dependent growth assays. When both MCF 7 control and MCF 7 TamR cells were treated with 4 OH Tam at increasing concentrations from 10 7 M to 10 5 M, the survival ratios showed marked differences between the two cell lines. selleck chemicals For instance, at 100 nM 4 OH Tam, MCF 7 TamR cells maintained a 90% survival ratio, compared to 60% for the tamoxifen sensitive MCF 7 con trol cells. At 4 uM, the ratio dramatically decreased to 30% for the control cells but remained around 70% for MCF 7 TamR. This trend continued until 4 OH Tam concentration reached Inhibitors,Modulators,Libraries 10 uM where no cells survived from either cell line.
To further investigate the proliferative behavior of the resistant cell line clonogenic assays were also performed. MCF 7 control Inhibitors,Modulators,Libraries and MCF 7 TamR cells were each treated with vehicle or 100 nM 4 OH Tam. The prolif eration of tamoxifen sensitive MCF 7 cells was signifi cantly inhibited in the presence of 100 nM Inhibitors,Modulators,Libraries 4 OH Tam. In contrast, the MCF 7 TamR cells demon strated strong resistance to 4 OH Tam induced inhibition of colony formation. Shown in Figure 2B is a representa tive image of colony formation of the two cell lines treated with vehicle and 4 OH Tam, respectively. Proteomics data reveal extensive changes in expression associated with acquired tamoxifen resistance To increase the total number of proteins that can be identified and quantified in whole cell lysates, we used a gel free approach that relies on isobaric mass tag labeling for quantitative analysis and a combination of two dimensional HPLC separation and high resolution mass spectrometry for maximal peptide detection and identifi cation.
Indeed, this approach yielded a total of 2,128 identified and 2,088 quantified proteins, which represent five to six times more proteins than were analyzed by a 2D gel based approach used in our previous study. Of these proteins, over 1,200 were found Inhibitors,Modulators,Libraries to have statisti cally significant changes in expression in the tamoxifen resistant cell line. While this number appears high, it reflects the high con fidence in the analytical reproducibility because the P values were calculated from the three isobaric labels as analytical replicates for each cell line sample.
Thus, some of the smaller fold changes in protein expression, while statistically significant and accurately reflective of the relative Inhibitors,Modulators,Libraries protein quantities in the two cell lines, may not be biologically relevant selleck chemicals Ixazomib to acquired tamoxifen resistance. When the minimum fold change value was set at two times the standard deviation of all protein ratios in the control sample, the total number of significantly chan ged proteins was reduced to 629 with 364 up regulated and 265 down regulated.