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Introduction
Buckwheat, an important functional food planted at high latitudes and in colder climates, is a common dietary component in East Asian countries (1). It is high in protein, contains a number of amino acids and is regarded as a popular food globally, particularly in Asia. Buckwheat protein has high biological value due to its well-balanced amino acid composition and its high level of lysine (2,3). Protease inhibitors are widely distributed in nature and are found in numerous animals, plants and microorganisms. They are important in maintaining the balance of proteolytic enzymes in vivo. In addition, they regulate endogenous proteases during germination and protect plants against insects and microorganisms (4–6). Of note, the inhibitors have been found to exhibit anti-carcinogenic activities and act as cancer-preventive and anti-inflammatory agents (7–9). Protease inhibitors are capable of inducing the apoptosis of cancer cells in vitro and they have drawn attention as potential anti-cancer agents (10). The Bowman-Birk inhibitor family of proteins attained from soybeans are associated with anti-inflammatory and anti-carcinogenic activities (11), and are potentially relevant anti-tumor agents, particularly with regard to colon cancer (12).
Numerous lines of evidence have suggested that protease inhibitors may induce apoptosis in various tumor cell lines; however, the underlying mechanisms of their anti-tumor activity remain to be elucidated. Induction of tumor cell apoptosis is a common mechanism of action of cancer therapeutics (13,14). Caspase-3 is one of the key initiators of apoptosis via the mitochondrial pathway and an essential factor for the activation of the caspase cascade (15–17). Recent studies have revealed that the activation of caspase-9 also induced the activation of the caspase cascade, triggering apoptotic events and inducing cell apoptosis (15,18,19). In addition, another pathway associated with apoptosis is the extrinsic pathway, which is associated with death receptors, including Fas. Adaptor molecules are recruited to the receptors following Fas ligand binding to the Fas death receptor, initiating the program of apoptosis (16,20,21).
Previous studies by our group revealed that a trypsin inhibitor from buckwheat was able to markedly inhibit the proliferation of the IM-9 and K562 cell lines in vitro (22,23). In order to elucidate whether the recombinant buckwheat trypsin inhibitor (rBTI) has the same effect in vivo and which apoptotic pathway is activated following rBTI treatment, the...