Analysis of Protein Expression during Oxidative Stress in Breast Epithelial Cells Using a Stable Isotope Labeled Proteome Internal Standard

Normal cells undergo a variety of molecular and physiological changes upon malignant transformation, including their responses to environmental factors that induce oxidative stress. Understanding the molecular pathways regulating these changes would facilitate the development of novel cancer treatments and chemoprevention strategies. Differences in the oxidative stress response were investigated between nonmalignant (S-1) and malignant (T4-2) cell lines (both derived from the HMT-3522 breast epithelial cells) using proteomic approaches. A modification of the stable isotope labeling of amino acids in cell culture (SILAC) approach was employed in which a [13C,15N]-labeled proteome was prepared from both cells. Relative quantification of the proteome derived from the S-1 cells and the T4-2 cells was then conducted using a [13C,15N]-labeled proteome as the internal standard. Differentially expressed proteins that changed in a similar manner in both cell lines were mainly stress response proteins, including heat shock proteins, peroxiredoxins, and redox proteins. Proteins that showed significant change in expression level in only one the cell lines included cytoskeleton proteins and proteins implicated in cell cycle and apoptosis regulation. Fortilin was found to be significantly up regulated in the transformed T4-2 cells after H2O2 treatment but not in the parental S-1 cells. However, Ran/TC4 was up regulated by H2O2 in the nonmalignant breast epithelial cells but not in the malignant cells. These results suggest that the malignant T4-2 cells have acquired more resistance to H2O2-induced apoptosis than the nonmalignant S-1 cells. Keywords: oxidative stress • quantitative proteomics • stable isotope label • internal standard