Effects of an anticarcinogenic Bowman-Birk protease inhibitor on purified 20S proteasome and MCF-7 breast cancer cells.
Proteasome inhibitors have been described as an important target for cancer therapy due to their potential to regulate the ubiquitin-proteasome system in the degradation pathway of cellular proteins. Here, we reported the effects of a Bowman-Birk-type protease inhibitor, the Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI), on proteasome 20S in MCF-7 breast cancer cells and on catalytic activity of the purified 20S proteasome from horse erythrocytes, as well as the structural analysis of the BTCI-20S proteasome complex. In vitro experiments and confocal microscopy showed that BTCI readily crosses the membrane of the breast cancer cells and co-localizes with the proteasome in cytoplasm and mainly in nucleus. Indeed, as indicated by dynamic light scattering, BTCI and 20S proteasome form a stable complex at temperatures up to 55°C and at neutral and alkaline pHs. In complexed form, BTCI strongly inhibits the proteolytic chymotrypsin-, trypsin- and caspase-like activities of 20S proteasome, indicated by inhibition constants of 10(-7) M magnitude order. Besides other mechanisms, this feature can be associated with previously reported cytostatic and cytotoxic effects of BTCI in MCF-7 breast cancer cells by means of apoptosis.
Laboratory of Biophysics, Department of Cellular Biology, University of Brasília, Brasília, Brazil.
Larissa da Costa
Lda C
Souza
Laboratory of Microbiology Department of Cellular Biology, University of Brasília, Brasília, Brazil.
Ricardo
R
Camargo
Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, Brazil.
Marilene
M
Demasi
Laboratory of Pathogen-Host Interface, Department of Cellular Biology, University of Brasília, Brasília, Brazil.
Jaime Martins
JM
Santana
Laboratory of Microbiology Department of Cellular Biology, University of Brasília, Brasília, Brazil.
Cézar Martins
CM
de Sá
Laboratory of Biophysics, Department of Cellular Biology, University of Brasília, Brasília, Brazil.
Sonia Maria
SM
de Freitas
D000818:Animals; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D004912:Erythrocytes / Q000378:metabolism*; D007887:Fabaceae / Q000737:chemistry*; D005260:Female; D005455:Fluorescent Antibody Technique; D006736:Horses; D006801:Humans; D061986:MCF-7 Cells; D046912:Multiprotein Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000032:analysis / Q000378:metabolism*
D046912:Multiprotein Complexes; D014361:Trypsin Inhibitors; D002918:Chymotrypsin; D046988:Proteasome Endopeptidase Complex