The anti-proliferative effect of TI1B, a major Bowman–Birk isoinhibitor from pea (Pisum sativumL.), on HT29 colon cancer cells is mediated through protease inhibition

Abstract

Bowman -Birk inhibitors (BBI) from legumes, such as soyabean, pea, lentil and chickpea, are naturally occurring plant protease inhibitors which have potential health-promoting properties within the mammalian gastrointestinal tract. BBI can survive both acidic conditions and the action of proteolytic enzymes within the stomach and small intestine, permitting significant amounts to reach the large intestine in active form to exert their reported anti-carcinogenic and anti-inflammatory properties. In a previous study, we reported the ability of a recombinant form of TI1B (rTI1B), representing a major BBI isoinhibitor from pea, to influence negatively the growth of human colorectal adenocarcinoma HT29 cells in vitro. In the present study, we investigate if this effect is related directly to the intrinsic ability of BBI to inhibit serine proteases. rTI1B and a novel engineered mutant, having amino acid substitutions at the P 1 positions in the two inhibitory domains, were expressed in the yeast Pichia pastoris. The rTI1B proved to be active against trypsin and chymotrypsin, showing K i values at nanomolar concentrations, whereas the related mutant protein was inactive against both serine proteases. The proliferation of HT29 colon cancer cells was significantly affected by rTI1B in a dose-dependent manner (IC 50 ¼ 31 (SD 7) mM), whereas the inactive mutant did not show any significant effect on colon cancer cell growth. In addition, neither recombinant protein affected the growth of non-malignant colonic fibroblast CCD-18Co cells. These findings suggest that serine proteases should be considered as important targets in investigating the potential chemopreventive role of BBI during the early stages of colorectal carcinogenesis.Key words: Bowman -Birk inhibitors: Cell proliferation: Colorectal cancer cells: Pea: Protease inhibition: Serine proteases Colorectal cancer has become one of the leading causes of cancer-related deaths worldwide and is currently increasing in Western countries(1) ; consequently, much attention has been focused on preventive and therapeutic strategies. The beneficial effects of certain bioactive compounds, mainly present in fruits and vegetables, in preventing and treating colorectal cancer have become a major focus of research (2 -4) . Among these, Bowman-Birk inhibitors (BBI) from legumes, such as soyabean (Glycine max), pea (Pisum sativum), lentil (Lens culinaris) and chickpea (Cicer arietinum), have been shown to have potential in protecting against inflammatory disorders and cancer development within the mammalian gastrointestinal tract (5 -8) . BBI are naturally occurring protease inhibitors with the ability to inhibit specifically serine (predominantly trypsin-and chymotrypsin-like) proteases. BBI are extensively disulphide-linked within proteins and have been demonstrated to be structurally and functionally resistant to the challenges (acidic conditions and the action of proteolytic enzymes) of the gastrointestinal tract in vivo. The conformational rigidity of BBI linked to t...

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:…”

Antiproliferative Activity of Lavatera cashmeriana- Protease Inhibitors towards Human Cancer Cells

“…IC 50 values suggest that NCIH322 lung cancer cells are strongly inhibited by both inhibitors at lower concentrations in comparison to other cell lines. Their pharmacological effects may be mediated through inhibition of the protease activity and the subsequent modulation of the protease pro-survival signaling as LC-pi I and II strongly inhibited the in vitro activity of trypsin, elastase and chymotrypsin (Clemente et al, 2010), also TI1B, a major Bowman-Birk isoinhibitor from pea (Pisum sativum L.) on HT29 colon cancer cells (Clemente et al, 2012) and antitumor Protease Inhibitor from Mini-Black Soybean (Ye and Ng, 2011). These evidences have made the plant protease inhibitors to be considered as neutraceutical proteins, owing to prevention and treatment of cancer (de Mejia and Dia, 2010).…”

Bowman-Birk Inhibitors from Legumes: Utilisation in Disease Prevention and Therapy

“…Recently, the anti-proliferative effect of rTI1B, a major pea isoinhibitor expressed heterologously in Pichia pastoris, has been evaluated using colon cancer cells grown in vitro. Comparisons of the effects of rTI1B with those observed using a related synthetic mutant derivative, showed that the proliferation of HT29 colon cancer cells was inhibited significantly by rTI1B in a dose-dependent manner, whereas the mutant which lacked trypsin and chymotrypsin inhibitory activity did not show any significant effect on colon cancer cell growth (Figure 1) [68]. Although the molecular mechanism(s) of this chemopreventive activity remains unknown, the reported data indicate that both trypsin-and chymotrypsin-like serine proteases involved in carcinogenesis are likely primary targets for BBI.…”