Abstract
Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development. In this study we purified and characterized the apoptosis-inducing factors secreted by normal breast epithelial cells. Conditioned medium was concentrated by ultrafiltration and separated on reverse phase Sep-Pak C18 and HPLC. The proapoptotic activity of eluted fractions was tested on MCF-7 breast cancer cells, and nano-LC-nano-ESI-MS/MS allowed the identification of insulin-like growth factor-binding protein-3 (IGFBP-3) and maspin as the proapoptotic factors produced by normal breast epithelial cells. Western blot analysis of conditioned media confirmed the specific secretion of IGFBP-3 and maspin by normal cells but not by breast cancer cells. Immunodepletion of IGFBP-3 and maspin completely abolished the normal cell-induced apoptosis of cancer cells, and recombinant proteins reproduced the effect of normal cell-conditioned medium on apoptosis of breast cancer cells. Together our results indicated that normal breast epithelial cells can induce apoptosis of breast cancer cells through IGFBP-3 and maspin. These findings provide a molecular hypothesis for the long observed inhibitory effect of normal surrounding cells on breast cancer development.
Highlights
Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development
Mass spectrometry analysis together with immunodepletion assay showed insulin-like growth factorbinding protein-3 (IGFBP-3) and maspin to be the major apoptosis-inducing factors produced by NBECs
NBEC-conditioned Medium Induced Apoptosis of Breast Cancer Cells—MCF-7 breast cancer cells were cultured in serum-free medium in the presence of different dilutions of NBEC-conditioned medium
Summary
Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development. Each eluted fraction was tested for its apoptosis-inducing activity in MCF-7 breast cancer cells as described above. This confirmed that IGFBP-3 and maspin secreted by NBECs did induce apoptosis of MCF-7 breast cancer cells.
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