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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 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.

Lunasin is a unique 43 amino acid soy peptide that has been shown to be chemopreventive in mammalian cells and in a skin cancer mouse model in this work against oncogenes and chemical carcinogens. The observation that lunasin inhibits core histone acetylation led to the proposal of an epigenetic mechanism by which lunasin selectively kills cells that are being transformed by disrupting the dynamics of cellular histone acetylation-deacetylation when the transformation event is triggered by the inactivation of tumor suppressors that function via histone deacetylation. Here is reported for the first time the core histone H3- and H4-acetylation inhibitory properties of lunasin from different Korean soybean varieties used for various food purposes and from tissues of rats fed with lunasin-enriched soy (LES) to measure bioavailability. Lunasin was analyzed by immunostaining and inhibition of core histone acetylation by a non-radioactive histone acetyl transferase assay. Various amounts of lunasin are found in the soybean varieties, which correlated with the extent of inhibition of core histone acetylation. Both soy lunasin and synthetic lunasin inhibit core histone acetylation in a dose-dependent manner. Lunasin in LES is protected from in vitro digestion by pepsin. Lunasin extracted from blood and liver of rats fed with LES is intact and inhibits core histone acetylation.

Lunasin, a unique 43 amino acid, 4.8 kDa cancer-chemopreventive peptide initially reported in soybean and now found in barley and wheat, has been shown to be cancer-chemopreventive in mammalian cells and in a skin cancer mouse model against oncogenes and chemical carcinogens. To identify bioactive components in traditional herbal medicines and in search for new sources of lunasin, we report here the properties of lunasin from Solanum nigrum L. (SNL), a plant indigenous to northeast Asia. Lunasin was screened in the crude extracts of five varieties of the medicinal plants of Solanaceae origin and seven other major herbal plants. An in vitro digestion stability assay for measuring bioavailability was carried out on SNL crude protein and autoclaved SNL using pepsin and pancreatin. A nonradioactive histone acetyltransferase (HAT) assay and HAT activity colorimetric assay were used to measure the inhibition of core histone acetylation. The inhibitory effect of lunasin on the phosphorylation of retinoblastoma protein (Rb) was determined by immunoblotting against phospho-Rb. Lunasin isolated from autoclaved SNL inhibited core histone H3 and H4 acetylation, the activities of the HATs, and the phosphorylation of the Rb protein. Lunasin in the crude protein and in the autoclaved crude protein was very stable to pepsin and pancreatin in vitro digestion, while the synthetic pure lunasin was digested at 2 min after the reaction. We conclude that lunasin is a bioactive and bioavailable component in SNL and that consumption of SNL may play an important role in cancer prevention.

Lunasin is a unique 43-amino acid peptide that has been shown to be chemopreventive in mammalian cells and in a skin cancer mouse model against oncogenes and chemical carcinogens. In search for new sources of lunasin and to better understand the role of cereals in cancer prevention, we report here the properties of lunasin from rye. The stability and bioavailability were measured by in vitro digestibility assay using pepsin and pancreatin and feeding rats with lunasin-enriched rye (LER). Inhibition of histone acetyl transferase (HAT) and nuclear localization in mammalian cells were used to measure lunasin bioactivity. Lunasin is present in 15 out of 21 cultivars of rye analyzed. Lunasin present in rye crude protein preparation is stable to pepsin and pancreatin in in vitro digestion. The liver, kidney, and blood of rats fed LER show the presence of lunasin in Western blot. Lunasin extracted from these tissues inhibits the activities of HATs, confirming that the peptide is intact and bioactive. Lunasin purified from rye internalizes in the nuclei of mouse fibroblast cells. We conclude that lunasin in rye is bioavailable and bioactive and that consumption of rye may play an important role of cancer prevention in rye-consuming populations.

It has been previously demonstrated that lunasin is a novel and promising cancer preventive peptide from soybean. The Bowman-Birk protease inhibitor (BBI) and isoflavones are well-studied substances from soy. This study evaluated the levels and bioactivities of these three compounds as affected by stages of seed development and sprouting under light and dark conditions. BBI and lunasin appear at 7 and 6 weeks, respectively, after flowering and increase as the seed matures. Daidzein and genistein both decrease during seed maturation. During sprouting under light, BBI increases up to the 6th day and decreases thereafter, disappearing at the 9th day after soaking. Under dark conditions, BBI increases up to the 7th day after soaking and decreases thereafter, disappearing at the 10th day. Lunasin starts to decrease at 2 days after soaking and disappears completely at 7 days under light and dark conditions. Daidzein and genistein increase continuously during the 10 days of soaking, and both increase more in the dark than under light conditions. Protein extracts from early seed development (2-5 weeks after flowering) suppress cell viability to a greater degree than those from later stages (6-9 weeks). Inhibition of foci formation by protein extracts from later stages is greater than those from earlier stages. Lunasin and BBI suppress foci formation more than the isoflavones. Sprouting decreases lunasin and BBI contents but increases isoflavones. Protein extracts from early soaking times inhibit foci formation more and suppress cell viability less than those from later soaking times. Light and dark conditions have no influence on the bioactivities of protein extracts. These data are useful in the preparation of soy fractions enriched in lunasin, BBI, and isoflavones and in making dietary recommendations.

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