Differential proteomic analysis of a highly metastatic variant of human breast cancer cells using two-dimensional differential gel electrophoresis

Abstract

Distant metastasis represents the major lethal cause of breast cancer. To understand the molecular mechanisms of breast cancer metastasis and identify markers with metastatic potential, we established a highly metastatic variant of parental MDA-MB-231 cells (MDA-MB-231HM). Using two-dimensional electrophoresis (2-DE), we performed a proteomic comparison of the two kinds of cells. As much as 51 protein spots were differentially expressed between the selected variant and its parental counterpart in at least 3 experiments. Ten unique proteins were identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), and database searching software. Among them, nine proteins were up-regulated in MDA-MB-231HM cells, including Macrophage-capping protein (CapG), Galectin-1, Chloride intracellular channel protein 1, Endoplasmic reticulum protein ERp29 precursor, Stathmin-1 (STMN1), Isoform 1 of uridine-cytidine kinase 2(UCK2), Rho GDP-dissociation inhibitor 2 (ARHGDIB), isocitrate dehydrogenase [NADP] cytoplasmic (IDH1), and N-myc downstream regulated gene 1 (NDRG1) protein. Only transgelin-2 was down-regulated. Differential expression was confirmed for three proteins including CapG, STMN1, and transgelin-2 by Western blotting analysis. Transgelin-2 was chosen for further verification by immunohistochemistry. The results suggested that 2-DE would be an efficient way to screen the proteins responsible for specific biological function. Furthermore, the findings imply that different proteins may be involved in the metastatic process in breast carcinomas.

Fig. 1

Karyotype analysis of MDA-MB231HM cells by Giemsa chromosome banding stain (×100)

Fig. 2

a Representative microphotographs of invasive comparison of MDA-MB-231 cells and MDA-MB-231HM cells through respective Matrigel-coated transwell membranes (×200). b Invasive cell numbers per field of MDA-MB-231 cells and MDA-MB-231HM cells after 24 h

Fig. 3

a Representative microphotographs of pulmonary metastasis lesions from BALB/c-nu/nu athymic mice bearing MDA-MB-231 and MDA-MB-231HM tumors. (H.E., ×100). b Statistical analysis for the number of lung metastasis deposits from the athymic mice bearing the both tumors after 5 weeks, p < 0.05

Fig. 4

Representative silver-stained 2-DE patterns of total cellular extracts from MDA-MB-231 (left) and MDA-MB-231HM (right) cells. Total proteins (170 μg) were applied to pH 3–10 nonlinear IPG strips (13 cm), and with 12% constant vertical SDS-PAGE as the second dimension. Gels were visualized by silver staining, and the resulting images were analyzed by PD Quest software. Protein Spots marked with a number indicate 10 proteins identified whose abundance changes when comparing MDA-MB-231 versus MDA-MB-231HM

Fig. 5

Differentially expressed protein spots in enlarged images. The pictures marked 1C–10C and 1 V–10 V correspond to the marked protein spots 1–10 in images of MDA-MB-231 (C) and MDA-MB-231HM (V) in Fig. 4. The differentially expressed protein spots are arrowed and marked with numbers accordingly

Fig. 6

Mass spectrometry of the peptides derived from spots 1(CapG). Each spot was removed from the gel, and in-gel digestion was performed using trypsin. The digested peptides were analyzed by mass spectrometry using a 4700 proteomics analyzer. The results of MS (a) and MS/MS (b, c) analysis are shown

Fig. 7

LC-ESI-MS/MS result for spot 6 (STMN1). a Spot 6 total ion chromatogram; b peptide MS map for spot 6 at 10.95 min; c MS/MS map for peptide 584.11

Fig. 8

a Representative immunodetection of CAPG, transgelin-2, STMN1. Total proteins (60 μg) extracted from MDA-MB-231 and MDA-MB-231 HM cells were separated by 12% SDS-PAGE gels and transferred to PDVF membrane. Immunoblotting was performed by rabbit antihuman polyclonal antibodies against CAPG, transgelin-2, and STMN1, and detected by ECL. Equal protein loading was evidenced by detection of GAPDH level using a monoclonal mouse antihuman GAPDH antibody. b The relative expressions of transgelin-2, CAPG, STMN1 protein in above two groups of cells was normalized to the signal intensity of GAPDH as an internal control

Fig. 9

Representative photographs of immunohistochemical analysis of transgelin-2 protein expression in human breast. All tissues were counterstained with hematoxylin and viewed by light microscopy (×100). Transgelin-2 expression in lymph node-negative tissues (a) and lymph node-positive tissues (b)