Secretome-based identification of tumorigenic and metastatic breast cancer biomarkers

Abstract

Breast cancer is one of the most common cancers in the world, and it is the leading cancer-related death in women. There is a need in discovery of prospective biomarkers in early detection for breast cancer. In this experiment, we applied proteomic technology 2D-DIGE coupled with MALDI-TOF mass spectrometry to investigate the proteome profile of MCF-10A human normal mammary cell and MCF-7、MDA-MB-231 human breast cancer cells under serum-free medium treatment, and hopefully screen out any potent biomarkers. Firstly, we incubated MCF-10A human mammary cell and MCF-7、MDA-MB-231 breast cancer cells in serum-free medium for 30 hours, and then collected the medium which is the source where proteins are purified from. Secondly, we utilized 2D-DIGE technique to investigate the protein expression profiles of each three cell lines under conditioned medium treatment. With the aid of the software Decyder and comassie blue stain, we are able to screen and pick out 175 protein spots that were differentially expressed above 1.5 fold. Thirdly, 90 proteins from these differentially expressed proteins are identified by MALDI-TOF mass spectrometry. Moreover, Western blot validation results proved this strategy as a reliable tool for searching biomarker candidates. Last but not the least, we are interested in those proteins that are related to tumor physiology such as tumor cell growth、tumor progression、cell survival and choose cathepsin D and flavin reductase for further biofunctional assay. We treated MCF-10A、MCF-7 and MDA-MB-231 with the protein inhibitors of cathepsin D and flavin reductase ,respectively. Observing by MTT assay, we found that there is a slightly different survival rate between the cells at low concentration of protein inhibitors, but death to all cells at a certain higher concentration, suggesting that both protein inhibitors and cathepsin D and flavin reductase may not be suitable as a direct therapeutic target for breast cancer.