Identification of 14-3-3σ as a Contributor to Drug Resistance in Human Breast Cancer Cells Using Functional Proteomic Analysis

Yang Liu, Hailan Liu, Baoguang Han, Jian-Ting Zhang
2006 Cancer Research  
Multidrug resistance (MDR) is a major obstacle to successful cancer treatment. To understand the mechanism of MDR, many cancer cell lines have been established, and various mechanisms of resistance, such as ATP-binding cassette (ABC) transporter-mediated drug efflux, have been discovered. Previously, a MDR cell line MCF7/AdVp3000 was selected from breast cancer cell line MCF7 against Adriamycin, and overexpression of ABCG2 was thought to cause MDR in this derivative cell line. However, ectopic
more » ... verexpression of ABCG2 in MCF7 cells could not explain the extremely high drug resistance level of the selected MCF7/AdVp3000 cells. We hypothesized that MCF7/AdVp3000 cells must have other resistance mechanisms selected by Adriamycin. To test this hypothesis, we compared the global protein profiles between MCF7 and MCF7/AdVp3000 cells. Following two-dimensional gel electrophoresis and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry analysis, 17 protein spots with differential levels between the two cell lines were identified. Although 14-3-3S, keratin 18, keratin 19, ATP synthase B, protein disulfide isomerase, heat shock protein 27, cathepsin D, triose-phosphate isomerase, peroxiredoxin 6, and electron transfer flavoprotein were increased, nm23/H1, peroxiredoxin 2, nucleophosmin 1/B23, and inorganic pyrophosphatase were decreased in MCF7/AdVp3000 cells. The differential levels of these proteins were validated using Western blot. Furthermore, functional validation showed that the elevated 14-3-3S expression contributes considerably to the observed drug resistance in MCF7/AdVp3000 cells. We, thus, conclude that these proteins likely contribute to the resistance selected in the MCF7/AdVp3000 cells, and their altered expression in tumors may cause clinical resistance to chemotherapy. (Cancer Res 2006; 66(6): 3248-55) Materials and Methods Materials. All electrophoresis reagents, precast SDS-PAGE slab gels, immobilized pH gradient strips, iodoacetamide, and polyvinylidene difluoride (PVDF) membranes were purchased from Bio-Rad (Hercules, CA). Adriamycin, mitoxantrone, verapamil, DTT, acetonitrile, and a-cyano-4-hydroxycinnamic acid were from Sigma (St. Louis, MO). Modified trypsin Requests for reprints: Jian-Ting Zhang,
doi:10.1158/0008-5472.can-05-3801 pmid:16540677 fatcat:tz6s5yqdczd7bp3ilw676ainb4