Comparative proteomics study of proteins involved in induction of higher rates of cell death in mitoxantrone-resistant breast cancer cells MCF-7/MX exposed to TNF-α

Abstract

Objectives: Resistance to medications is one of the main complications in chemotherapy of cancer. It has been shown that some multidrug resistant cancer cells indicate more sensitivity against cytotoxic effects of TNF-α compared to their parental cells. Our previous findings indicated vulnerability of the mitoxantrone-resistant breast cancer cells MCF-7/MX to cell death induced by TNF-α compared to the parent cells MCF-7. In this study, we performed a comparative proteomics analysis for identification of proteins involved in induction of higher susceptibility of MCF-7/MX cells to cytotoxic effect of TNF-α.

Materials and methods: Intensity of protein spots in 2D gel electrophoresis profiles of MCF-7 and MCF-7/MX cells were compared with Image Master Platinum 6.0 software. Selected differential protein-spots were identified with MALDI-TOF/TOF mass spectrometry and database searching. Pathway analyses of identified proteins were performed using PANTHER, KEGG PATHWAY, Gene MANIA and STRING databases. Western blot was performed for confirmation of the proteomics results.

Results: Our results indicated that 48 hr exposure to TNF-α induced 87% death in MCF-7/MX cells compared to 19% death in MCF-7 cells. Forty landmarks per 2D gel electrophoresis were matched by Image Master Software. Six proteins were identified with mass spectrometry. Western blot showed that 14-3-3γ and p53 proteins were expressed higher in MCF-7/MX cells treated with TNF-α compared to MCF-7 cells treated with TNF-α.

Conclusion: Our results showed that 14-3-3 γ, prohibitin, peroxiredoxin 2 and P53 proteins which were expressed differentially in MCF-7/MX cells treated with TNF-α may involve in the induction of higher rates of cell death in these cells compared to TNF-α-treated MCF-7 cells.

Keywords: 14-3-3 γ; MCF-7/MX cells; Mitoxantrone; Multidrug resistance; TNF-α.

Figure 1

Phase contrast microscope imaging after 48 hr treatment with 50 ng/ml TNF-α

Figure 2

Assessment of the cell viability status by flow cytometry

Figure 3

2D gel images. A and C are 2D gels colloidal coomassie blue of TNF-α-treated MCF-7 cells. B and D are 2D gels colloidal coomassie blue of TNF-α-treated MCF-7/MX cells. Six differentially expressed protein spots were identified by MALDI-TOF/TOF and MASCOT software

Figure 4

PANTHER classification of six identified proteins

Figure 5

Protein-protein interaction networks between the identified proteins and other proteins. Each protein is identified by a circle. Each color in circle is related to a function

Figure 6

A) Maps of interaction among selected protein species. Nodes and different line colors represent proteins and types of evidence among them respectively. B) Protein–protein interaction maps of selected protein species when p53 was added as a coordinator agent. Interactions between YWHAG, PHB and p53 play important role in the biological pathways including regulated cell death and cell cycle contro

Figure 7.

Comparison of 14-3-3γ and p53 proteins expression

Figure 8

TNF-α signaling pathway based on 14-3-3γ protein function. As mentioned in the text, exposure to TNF-α can lead to overexpression of 14-3-3γ protein in MCF-7/MX cells. This protein interacts with many proteins such as PKC and p53. 14-3-3γ acts as an inhibitor for PKC activity leading to blocking of PHB phosphorylation. Inhibition of PHB phosphorylation elevates mitochondrial permeability and facilitates regulated cell death procedures. 14-3-3γ also elevates stability and activity of p53 via inhibition of AKT phosphorylation which might be another mechanism connecting 14-3-3γ to cytotoxic effects of TNF-α in MCF-7/MX cells