Lunasin is a novel peptide originally identified in soybean that suppresses chemical carcinogen-induced transformation in mammalian cells and skin carcinogenesis in mice. Since the lunasin gene was cloned from soybean and the chemically synthesized form of the lunasin peptide has been used in experiments conducted so far, the isolation of lunasin from other natural sources and testing of its biological properties have not been carried out. We report here the isolation, purification, and biological assay of lunasin from barley, a newly found rich source of the peptide. The identity of lunasin was established by Western blot analysis and mass spectrometric peptide mapping of the in-gel tryptic digest of the putative protein band. Lunasin was partially purified with anion exchange and immunoaffinity chromatography. The crude and partially purified lunasin from barley suppressed colony formation in stably ras-transfected mouse fibroblast cells induced with IPTG. These fractions also inhibited histone acetylation in mouse fibroblast NIH 3T3 and human breast MCF-7 cells in the presence of the histone deacetylase inhibitor sodium butyrate.

Lunasin, a novel and promising chemopreventive compound isolated from soybean cotyledon, is a 43-amino acid peptide that contains a -RGD-cell adhesion motif followed by 8 aspartic acid residues at the carboxyl end and a structurally conserved helix region. We showed previously that lunasin peptide applied exogenously reduces foci formation in mouse fibroblast cells treated with chemical carcinogens and inhibits skin tumorigenesis induced by chemical carcinogens in mice when applied topically. In this study, lunasin peptide applied to cell culture suppresses foci formation in E1A-transfected mouse fibroblast NIH 3T3 cells. Within 18 h of exogenous application, lunasin internalizes into the cell and localizes in the nucleus. In an initial study of genes affected by lunasin, the peptide increases p21 protein levels fivefold in cells transfected with E1A but not in untransfected cells. In contrast to its inhibitory effects on cell transformation, lunasin has no effect on growth of imicroMortalized (nontumorigenc) and established cancer cells. This is the first report that lunasin suppresses transformation of mamicroMalian cells induced by an oncogene (E1A) in addition to chemical carcinogens.

Lunasin is a novel and promising chemopreventive peptide from soybean. We have shown previously that lunasin suppresses transformation of mammalian cells caused by chemical carcinogens and inhibits skin carcinogenesis in mice when applied topically. Although the lunasin gene was cloned from soybean, all experiments carried out so far in our lab have used synthetic lunasin and therefore there is no detailed description of natural lunasin isolated from soybean. We report here the first characterization of soybean lunasin that includes definitive identification by mass peptide mapping, partial purification, and measurement of bioactivities of the various purified fractions and protein expression in the developing seed. The identity of lunasin in the seed extracts was established by Western blot analysis and mass spectrometric peptide mapping. All lunasin fractions partially purified by anion exchange and immunoaffinity column chromatography suppress colony formation induced by the ras-oncogene and inhibit core H3-histone acetylation. During seed development, lunasin peptide appears 5 weeks after flowering and persists in the mature seed. Western blot analysis of different soybean varieties and commercially available soy proteins shows the presence of the peptide in varying amounts. These results demonstrate the feasibility of producing large quantities of natural lunasin from soybean for animal and human studies.