Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 2014; 18: 2070-2075 Lunasin as a promising health-beneficial peptide

J. LIU 1, S.-H. JIA 1, M. KIRBERGER 2, N. CHEN 1

1College of Health Science, Wuhan Sports University, Wuhan, China 2Department of Natural Sciences, Clayton State University, Morrow, GA, USA

Jun Liu and Shaohui Jia contributed equally to this work

Abstract. – Lunasin, a 43 amino acid characterized as a small polypeptide with poly- polypeptide originally isolated from , is known to produce multiple health benefits, in - Asp (D) residues located at the carboxyl termi - cluding anti-hypertension, antioxidant activity, nus , and was subsequently found in other beans, prevention or therapy, anti-inflamma - grains and herbal plants , including , , tion, hypocholesterolemic activity, anti-obesity , sunberry, wonderberry, bladder-cherry , jim - and immunomodulation. These effects are be - son weed , Solanum nigrum L ., amaranth and trit - lieved to be due to its unique structure that in - icale , at concentrations ranging from 0.013 to cludes a putative helical region, an Arg-Gly-Asp 4 (RGD) motif and an Asp-rich carboxyl terminus. 70.5 mg protein lunasin/g of protein (Table I ) . The focus of this article is to summarize the Since then, lunasin has been the subject of sever - discovery, characterization and biological activ - al investigations, including a very important ities of lunasin, which will provide a reference study on the cloning of the soybean lunasin gene for the future development and utilization of lu - and subsequent transfection into mammalian nasin, and a basis for exploring the underlying mechanisms of these health-beneficial func - cells, at the California University at Berkeley tions. (USA), which led to the discovery that the lu - nasin gene can disrupt mitosis and induce chro - Key Words: mosome breakage , ultimately leading to cell Lunasin, Anticancer, Anti-, Soybean apoptosis 5. peptide.

Characterization of Lunasin Discovery and Resources of Lunasin Structural Characteristics There is a growing body of research suggest - Lunasin is a unique peptide composed of 43 ing that the consumption of soybean products can amino acid residues (sequence SKWQHQQD - reduce the incidence of osteoporosis and some SCRKQKQGVNLTPCEKHIMEKIQ - chronic diseases, especially cardiovascular dis - GRGDDDDDDDDD ) ( Figure 1 ) with a molecu - eases and 1. Consumption of soybean lar weight of 5.5 kDa. This peptide contains 8 products is also associated with lower mortality negatively-charged Asp (D) residues at the car - rates from prostate, breast, colon, and endometri - boxyl terminus, thus providing a histone -binding al cancers 2. Therefore, the modification of nutri - property that allows it to act as a potent inhibitor tional habits (e.g., soybean consumption) is be - of positively-charged H3 and H4 histone acetyla - coming increasingly interesting as a means of tion 6. The poly-D chain is immediately preceded disease prevention and modulatory therapy. Pre - in the sequence by a cell adhesion motif Arg- vious studies have revealed that soybean contains Gly-Asp (RGD) that is responsible for the attach - a variety of phytochemicals including protease ment of lunasin to the extracellular matrix , and a inhibitors, inositol hexaphosphate (), putative helical region (EKHIMEIQG ) which is b-sitosterol, saponins, and isoflavones 3. structurally homologous to a conserved region Lunasin was first isolated in 1987 at Niigata found in chromatin-binding proteins 7, suggesting University School of Medicine in Japan, during that the helical region may target lunasin to core the screening of protease inhibitors from soybean histones 8. This putative helical region may func - seeds , and was subsequently identified as a tion to enhance binding capability to the core his - promising anticancer candidate. It was initially tone. The RGD motif can also facilitate internal -

2070 Corresponding Author: Ning Chen, Ph.D; e-mail: [email protected] Lunasin as a promising health-beneficial peptide

Figure 1. Amino acid sequences and functions of peptide fragments from lu - nasin. The function of the fragment spanning residues 1 -22 remains to be defined; the fragment spanning residues 23 -31 is responsible for chro - matin-targeting; the Arg-Gly-Asp motif (amino acid residues 32 -34) is respon - sible for peptide internalization into the cell nucleus; the polyaspartyl end (amino acid residues 35 -43) is respon - sible for its direct binding to chromatin.

ization of lunasin into mammalian cells within Physiological Characteristics several minutes , followed by localization in the Since lunasin is a peptide, it is important to nucleus within a few hours 8. According to recent understand the degradation, absorption, translo - studies, the cell adhesion function of the RGD cation and distribution of lunasin in target tissues motif may be cell-line specific, with adhesion or organs subsequent to oral administration. observed in C3H cells, but not in NIH3T3 cells . Therefore, the bioavailability of lunasin has been The RGD motif can induce cell apoptosis in dif - explored in both in vitro and in vivo studies. Pre - ferent cell lines via a caspase-dependent mecha - liminary studies regarding bioavailability of lu - nism 9. In addition, lunasin may also be involved nasin in mice and rats supplied with lunasin-en - in cellular growth and proliferation, cellular riched soybean protein have revealed that 35% of function maintenance , and cell -cell signaling and ingested lunasin is distributed to the target tissues interactions 10 . Based on our current knowledge, and organs in an intact and active form 11,12 . Simi - the major functions of lunasin appear to reside in larly, a study on the bioavailability of lunasin in a the poly-D chain, RGD motif and putative helical sample from adult human males indicated that region structures, while functions associated with 4.5% of the ingested lunasin was distributed in the other amino acid residues or structures in lu - the plasma in the intact or active form of the soy - nasin remain undefined . Further research will be bean protein 13 . The high capacity of lunasin to required to determine whether smaller peptides avoid degradation by gastrointestinal and serum that include these functional domains from lu - proteinases or peptidases, and retain its bioactive nasin will be capable of similar health-promoting form during the translocation process to blood or therapeutic functions. and other organs , makes lunasin a promising can -

Table I. Lunasin content in soybean, grains and medicinal plants.

Plant (Latin name) Plant (General name) Lunasin content Lunasin content (mg/g protein) (mg/g seed)

Triticum aestivum Wheat 0.2-0.3 Hordeum vugare L. Barley 5.9-8.7 0.01-0.02 Glycine max 5 Soybean 4.4-70.5 0.5-8.1 Amaranthus hypochondriacus Amaranth 9.5-12.1 Physalis alkekengi var. francheti Bladder-cherry 17.0 0.1 Solanum lyratum Hyyodori-jogo 22.3 0.4 Solanum nigrum L. Sunberry 36.4 1.8 Datura starmonium Jimson weed 10.3 0.3

2071 J. Liu, S.-H. Jia, M. Kirberger, N. Chen didate for the preventive treatment of cancers in mination of the inhibitory activity of linoleic acid vivo 14 . In addition, circular dichroism analysis re - oxidation and scavenging capacity of ABTS + free veals that lunasin exhibits high thermostability radicals . Lunasin appears to exhibit potent ABTS within the temperature range 25-100° C with no scavenging activity, with TEAC values of 1.41 17 apparent change in either its secondary structure and 1.90 µM, respectively . or its bioavailability 15 . Anti-inflammation Lunasin also appears to inhibit the release of Preparation of Lunasin pro-inflammatory cytokines such as tumor necro - sis factor- α (TNF- α) and interleukin -6 ( IL-6) in Current methods for the preparation of lunasin LPS-stimulated RAW264.7 cells, without im - include isolation and purification from natural pacting cell viability 17 . Its corresponding action products, prokaryotic expression and chemical mechanisms are strongly correlated with reduced synthesis. Jeong et al 12 isolated lunasin from inflammation in LPS-stimulated RAW 264.7 wheat seeds at different growth stages by extrac - macrophages , induced by lunasin through inhibi - tion using MPBS buffer supplemented with fresh tion of the nuclear factor kappa B (NF-kB) path - protease inhibitor and partial purification, and way 18 . Similarly, the treatment of RAW 264.7 identified the product using mass peptide map - macrophage with 100 µM lunasin apparently re - ping. The isolated lunasin is evaluated by de - duces the production of NO and prostaglandin terming the bioactivity of the various purified frac - E2 (PGE2 ), as well as reducing the expression of tions and intact protein. Vermont et al 26 applied a inducible nitric oxide synthase (iNOS ) and cy - method that utilized the sequential application of clooxygenase-2 (COX-2 ). anion-exchange chromatography, ultrafiltration, and reversed-phase chromatography 15 . Anticancer The lunasin gene can be synthesized by over - Lunasin, as a novel chemopreventive peptide lapping extension polymerase chain reaction derived from soybean, also functions to suppress (PCR) and expression in E. coli BL21 (DE3) chemical carcinogens and viral oncogene-in - with the use of vector pET29a. The recombinant duced transformation of mammalian cells , and to lunasin containing His-tag at the C-terminus can inhibit carcinogen-induced skin tumorigenesis in be expressed in soluble form so that it can be pu - mice . The addition of 125 nM lunasin can reduce rified by immobilized metal affinity chromatog - transformation efficiency in dimethylbenzan - raphy. The result of in vitro bioassays has re - thracene (DMBA )-treated C3H cells by 80% , vealed that the purified recombinant lunasin can while 3-methylcholantrene (MCA )-treated C3H inhibit histone acetylation. Recombinant lunasin cells treated with 10 nM lunasin for 24 h exhibit - also inhibits the release of pro-inflammatory cy - ed a 69% reduction in transformation efficiency . tokines (tumor necrosis factor- α [TNF- α] and in - Purified lunasin at concentrations of 50, 100, 250 terleukin-1 β), and the production of nitric oxide 500 and 1000 nM can inhibit yGCN5 activity by (NO) 15 . Additionally, lunasin fused with a hexa - 3, 13, 27, 48 and 65%, respectively 8,11 . Combina - histidine tag and tobacco etch virus protease site torial application of anacardic acid and lunasin allows for the protease-mediated release of native can result in the arrest of the cell cycle in S- lunasin, which can be more effectively analyzed phase , and apoptosis induction at a higher level , using the tobacco etch virus 16 . Moreover, lunasin and these results are not observed with applica - has also recently been synthesized using a solid tions of each compound individually . Additional - phase peptide synthesis strategy. ly , the combined application of both compounds produces synergistic inhibition of ERBB2, AKT1, JUN and RAF1 signaling genes, which Biological Activity of Lunasin are responsible for the growth of breast cancer cells and resistance to apoptosis when up-regu - Anti-oxidant or Anti-free Radical Capacity lated . Moreover, lunasin inhibits cell prolifera - A previous investigation by Hernández-Ledes - tion in a dose-dependent manner 6,19,20 . Histologi - ma et al 23 demonstrated that lunasin can reduce cal staining of samples collected from lunasin - the production of reactive oxygen species (ROS) treated tumors has revealed that destructive re - in lipolysaccharide (LPS )-induced macrophages gions of tumors are replaced by apoptotic and in a dose-dependent manner , based on the deter - necrotic cells , thus demonstrating that lunasin

2072 Lunasin as a promising health-beneficial peptide treatment also results in a significant reduction of cell adhesion motif, co -localizes with hy - cell proliferation and obvious apoptosis induction poacetylated chromatin, binds preferentially to in MDA-MB-231 tumors , although these effects deacetylated histone H4 in vitro and inhibits the are not observed in tumors from Bowman-Birk acetylation of histone H3 and H4 in vivo in the inhibitor (BBI )-treated mice. Together , these presence of histone deacetylase inhibitors 8. Lu - findings indicate that lunasin acts as a preventive nasin can also up -regulate the expression of agent for cancers in vivo , and that BBI prevents genes directly or indirectly involved in chemo - lunasin from digestion, thereby increasing its prevention in non -tumorigenic prostate epithelial bioavailability . Pure lunasin isolated from Bow - RWPE-1 cells but not in tumorigenic RWPE-2 man-Birk inhibitor concentrate (BBIC ) at 100 cells, and can enter both RWPE-1 and RWPE-2 nM can reduce foci formation by 73%, which is cells in a dose-dependent manner and co -local - identical to results observed using 100 nM syn - ize along the inner edge of the nuclear mem - thetic lunasin, while pure BBI isolated from the brane, a region known to be the site of actively BBIC at 100 nM decreases foci formation by transcribed chromatin 26,27 . It can also bind tightly 60%. Thus, lunasin is 18% more effective than and specifically to H4 in vitro to mask the acety - BBI at equivalent concentrations 21 . lation of H4K8 while enhancing the acetylation Lunasin has been found to inhibit cell prolif - of H4K16 , and can hyperacetylate H4K16 in vi - eration, arrest cell cycle in the S phase , and vo at the 5 ’ end of the pro-apoptotic gene down-regulate both mRNA levels of CDK2, THBS1 in RWPE-1 cells , but not in RWPE-2 CDK4, CDC25A, caspase 8, Ets2, Myc, Erbb2, cells. It is hypothesized that H4K16 acetylation AKT1, PIK3R1, FOS and JUN signaling genes in chromatin destabilizes electrostatic interac - in MDA-MB-231 cells , and the expression of tions between adjacent nucleosomes , thus im - Bcl-2 7,14,22 . Research also suggests that lunasin proving the accessibility of the basal transcrip - can reduce protein levels of cyclin D1, cyclin tion apparatus to the promoter 28-31 . D3, CDK4 and CDK6 in a dose-dependent man - ner in breast cancer cells , and down-regulate Bax and caspase-3 in HT-29 colon cancer Conclusions cells 7,23 . In MCF-7 cells, lunasin may up-regu - late the tumor suppressor phosphatase and tensin Lunasin contains multiple functional sites, in - homolog deleted in chromosome ten (PTEN). cluding a putative helical region, an RGD motif Lunasin-induced cellular apoptosis is similar to and a poly-D chain. The polyfunctional nature of apoptosis induced by in that both are lunasin makes it an attractive peptide for antioxi - mediated by PTEN, although the lunasin mecha - dant and radical-scavenging, and both anti-in - nism is p53-independent. Additionally , both lu - flammatory and anti -cancer functions . However , nasin and genistein effect the promotion of E- further investigation will be required to deter - cadherin and beta-catenin non -nuclear localiza - mine if shorter or smaller peptides containing tion , although lunasin does not appear to have an these lunasin functional domains can exhibit sim - impact on the oncogene Wnt1 in HC11 cells 24 . ilar health-promoting or therapeutic functions. Similarly, lunasin can slow the proliferation of Additional research will also be needed to deter - epidermal cells in mouse skin in both the ab - mine the underlying mechanisms of lunasin, par - sence and presence of 7,12-dimethylbenz(a)an - ticularly with respect to other potential therapeu - thracene (DMBA ). Lunasin applied topically at tic applications , such as the ability to inhibit in - 250 mg/week suppresses skin papilloma forma - flammation-related cancer progression or tion in SENCAR (sensitivity to carcinogenesis) rheumatoid arthritis . mice treated with DMBA and tetradecanoylphor - bol-13-acetate by 70% when compared with the ––––––––––––––––– –– control 25 . Lunasin has also demonstrated the Acknowledgements ability to significantly reduce keratinocyte pro - This work is financially supported by Chutian Scholar Pro - liferation in both normal and carcinogen-treated gram in Hubei Province and the Innovation Program from SENCAR mice 22 . When RWPE-2 cells are treat - Wuhan Sports University by NC. ed with 50 mM lunasin for 24 h, lunasin im - munofluorescent signals can be visualized in –––––––––––––––– –-– –– both the cytoplasm and the nucleus . Synthesized Conflict of Interest lunasin internalizes in the cell through the RGD The Authors declare that there are no conflicts of interest.

2073 J. Liu, S.-H. Jia, M. Kirberger, N. Chen

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