Effects of an Anticarcinogenic Bowman-Birk Protease Inhibitor on Purified 20S Proteasome and MCF-7 Breast Cancer Cells

Abstract

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.

Oligomerization affects the kinetics and thermodynamics of the interaction of a Bowman-Birk inhibitor with proteases

“…Moreover, the stoichiometry, kinetics and thermodynamics of the BTCI complexation with -CT are consistently influenced by the increment in protein concentrations from the nanomolar to the micromolar range [13,14,26]. Our study indicates that secondary interactions should play an important role in the kinetic and thermodynamic parameters in protein interactions in certain experimental setups.…”

“…It presents rich biotechnological potential as an anticarcinogenic agent, as a guanylin-enhancing natriuresis and renal functional response in rats, as a bradykinin-potentiating molecule, and as an insecticide, inhibiting the growth of larvae with agricultural importance [7][8][9][10][11][12].…”

Soybean-derived Bowman-Birk inhibitor (BBI) blocks HIV entry into macrophages

“…In the present study, we identified that BBI, a natural product in soybean, is a potent entry inhibitor of HIV infection of macrophages. BBI has been shown to have several therapeutic activities, including anti-inflammation (Dai et al, 2012; Safavi and Rostami, 2012) and anti-cancer (Souza Lda et al, 2014). Several studies (Ye et al, 2001; Ye and Ng, 2009; Prasad et al, 2010) showed that BBI could inhibit HIV reverse transcriptase in cell-free systems, with the IC 50 of 49 μM (equal to 387 μg/ml) (Ye et al, 2001).…”

Plant Protease Inhibitors in Therapeutics-Focus on Cancer Therapy

“…Many of those naturally found PIs were further characterized from different plant species which mainly included trypsin from serine protease group which have been tested for various diseases (Richardson, 1991; Tamir et al, 1996; Majumdar, 2013). This review explains about PIs of all earlier reported plant species that have been used as therapeutic agents and tested against different diseases and human disorders (Table 1; Murugesan et al, 2001; Neuhof et al, 2003; Troncoso et al, 2003; Kobayashi et al, 2004; Lanza et al, 2004; Clemente et al, 2005, 2012; Kim et al, 2005; Suzuki et al, 2005; Capaldi et al, 2007; Banerjee et al, 2008; Tochi et al, 2008; Caccialupi et al, 2010; Hsieh et al, 2010; Joanitti et al, 2010; García-Gasca et al, 2012; Magee et al, 2012; de Paula et al, 2012a; Borodin et al, 2013; Ferreira et al, 2013; Rakashanda et al, 2013b; Clemente and Arques, 2014; Souza et al, 2014) and future scope to search for new species, which are as follows:…”

“…The Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI) isolated and purified from Vigna unguiculata (Cowpea) seeds is a natural PPI, and it belongs to the BBI family with two different and independent reactive sites for trypsin (Lys26) and chymotrypsin (Phe53) (Souza et al, 2014). BTCI is a globular protein comprising 83 amino acid residues with seven disulfide bonds and molecular weight of 9.1 kDa (Barbosa et al, 2007).…”