1. Lunasin Peptide Upregulates Thrombospondin 1 (THBS1) Gene Expression in Non-Tumorigenic Prostate Epithelial Cells
Alfredo F. Galvez, Liping Huang, Mark M.J. Magbanua, Kevin Dawson, Raymond L. Rodriguez, Nutrition and Cancer (2011), In Press.
There is growing evidence supporting the role of epigenetic modifications in cancer formation as the result of dysregulation of gene expression, especially in the early stages of carcinogenesis. The objective of this study was to investigate the chemopreventive properties of lunasin at the molecular and cellular levels by examining gene expression patterns and the associated epigenetic changes in lunasin treated tumorgenic and non-tumorgenic prostate epithelial cell. The results reported may provide a molecular mechanism to explain the association between higher soy consumption and lower cancer risk.
2. Lunasin Potentiates the Effect of Oxaliplatin Preventing Outgrowth of Colon Cancer Metastasis, Binds to α5β1 Integrin and Suppresses FAK/ERK/NF-κB Signaling
Vermont P. Dia, Elvira Gonzalez de Mejia, Cancer Letters, (2011), 37 (34)
The effect of lunasin on colon cancer metastasis was studied using three human colon cancer cell lines in vitro and a liver metastasis model of colon cancer in vivo. Lunasin bond with α5β1 integrin and internalized into the nucleus of KM12L4 human colon cancer cells. Lunasin (10 μM) inhibited the activation of focal adhesion kinase (FAK) by 28, 39 and 60% in RKO, HCT-116 and KM12L4 human colon cancer cells, respectively. Lunasin caused an increase in the expression of the inhibitor of kappa B alpha (IκB-α), a decrease in nuclear p50 NF-κB and a reduction in the migration of cancer cells. Lunasin (4 mg/kg bw) inhibited metastasis and potentiated the effect of oxaliplatin by reducing the expression of proliferating cell nuclear antigen. Liver metastatic nodules were reduced from 28 (PBS) to 14 (lunasin, P = 0.047) while combination of lunasin and oxaliplatin to 5 (P = 0.004). The tumor burden was reduced from 0.13 (PBS) to 0.10 (lunasin, P = 0.039) to 0.04 (lunasin + oxaliplatin, P < 0.0001). Moreover, lunasin potentiated the effect of oxaliplatin in modifying expression of proteins involved in apoptosis and metastasis including Bax, Bcl-2, IKK-α and p-p65. Lunasin inhibited metastasis of human colon cancer cells by direct binding with α5β1 integrin suppressing FAK/ERK/NF-κB signaling, and potentiated the effect of oxaliplatin in preventing the outgrowth of metastasis.
3. Differential Expression of Thrombospondin (THBS1) in Tumorigenic and Nontumorigenic Prostate Epithelial Cells in Response to a Chromatin-Binding Soy Peptide
Alfredo F. Galvez, Liping Huang, Mark M. J. Magbanua, Kevin Dawson & Raymond L. Rodriguez, Nutrition and Cancer, (2011), 63 (4), 623-636.
The chemopreventive properties of the chromatin-binding soy peptide, lunasin, are well documented, but its mechanism of action is unclear. To elucidate the mechanism by which lunasin reduces tumor foci formation in cultured mammalian cells, nontumorigenic (RWPE-1) and tumorigenic (RWPE-2) human prostate epithelial cells were treated with lunasin followed by gene expression profiling and characterization of the chromatin acetylation status for certain chemopreventive genes. The genes HIF1A, PRKAR1A, TOB1, and THBS1 were upregulated by lunasin in RWPE-1 but not in RWPE-2 cells. Using histone acetyltransferase (HAT) assays with acid-extracted histones as templates, we showed that lunasin specifically inhibited H4K8 acetylation while enhanced H4K16 acetylation catalyzed by HAT enzymes p300, PCAF, and HAT1A. These results suggest a novel mechanism for lunasin-dependent upregulation of gene expression. Chromatin immunoprecipitation (ChIP) revealed hypoacetylation of H4K16 in RWPE-2 cells, specifically at the 5′ end of THBS1 containing a CpG island. Moreover, bisulfite PCR (BSP) and subsequent DNA sequencing indicated that this CpG island was hypomethylated in RWPE-1 but hypermethylated in RWPE-2 cells. Histone hypoacetylation and DNA hypermethylation in the 5′ region of THBS1 may explain the inability of lunasin to upregulate this gene in RWPE-2 cells.
4. Lunasin-Aspirin Combination Against NIH/3T3 Cells Transformation Induced by Chemical Carcinogens
Chia-Chien Hsieh, Blanca Hernandez-Ledesma, Ben O. de Lumen, Plant Foods for Human Nutrition, (2011), 66 (2), 107-113.
Carcinogenesis is a multistage process involving a number of molecular pathways sensitive to intervention. Chemoprevention is defined as the use of natural and/or synthetic substances to block, reverse, or retard the process of carcinogenesis. To achieve greater inhibitory effects on cancer cells, combination of two or more chemopreventive agents is commonly considered as a better preventive and/or therapeutic strategy. Lunasin is a promising cancer preventive peptide identified in soybean and other seeds. Its efficacy has been demonstrated by both in vitro and in vivo models. This peptide has been found to inhibit human breast cancer MDA-MB-231 cells proliferation, suppressing cell cycle progress and inducing cell apoptosis. Moreover, lunasin potentiates the effects on these cells of different synthetic and natural compounds, such as aspirin and anacardic acid. This study explored the role of lunasin, alone and in combination with aspirin and anacardic acid on cell proliferation and foci formation of transformed NIH/3T3 cells induced by chemical carcinogens 7,12-dimethylbenz[a]anthracene or 3-methylcholanthrene. The results revealed that lunasin, acting as a single agent, inhibits cell proliferation and foci formation. When combined with aspirin, these effects were significantly increased, indicating that this combination might be a promising strategy to prevent/treat cancer induced by chemical carcinogens.
5. Relationship between lunasin’s sequence and its inhibitory activity of histones H3 and H4 acetylation
Blanca Hernandez-Ledesma, Chia-Chien Hsieh, Ben O. de Lumen, Molecular Nutrition & Food Research, (2011) 55 (7), 989-998.
Scope: Dysfunction of histone acetyltransferases (HATs) or histone deacetylases (HDACs) involved in histones acetylation has been associated with cancer. Inhibitors of these enzymes are becoming potential targets for new therapies. Methods and Results: This study reports by Western-Blot analysis, that peptide lunasin is mainly an in vitro inhibitor of histone H4 acetylation by P300/cAMP-response element-binding protein (CBP)-associated factor (PCAF), with IC(50) values dependent on the lysine position sensitive to be acetylated (0.83 μM (H4-Lys 8), 1.27 μM (H4-Lys 12) and 0.40 μM (H4-Lys 5, 8, 12, 16)). Lunasin is also capable of inhibiting H3 acetylation (IC50 of 5.91 μM (H3-Lys 9) and 7.81 μM (H3-Lys 9, 14)). Studies on structure-activity relationship establish that lunasin's sequence are essential for inhibiting H4 acetylation whereas poly-D sequence is the main active sequence responsible for H3 acetylation inhibition. Lunasin also inhibits H3 and H4 acetylation and cell proliferation (IC(50) of 181μM) in breast cancer MDA-MB-231 cells. Moreover, this peptide decreases expression of cyclins and cyclin dependent kinases-4 and -6, implicated in cell cycle pathways. Conclusion: Results from this study demonstrates lunasin's role as modulator of histone acetylation and protein expression that might contribute on its chemopreventive properties against breast cancer.
6. Lunasin induces apoptosis and modifies the expression of genes associated with extracellular matrix and cell adhesion in human metastatic colon cancer cells
Vermont P. Dia, Elvira Gonzalez de Mejia, Molecular Nutrition & Food Research, (2011) 55 (4), 623-634.
SCOPE: Lunasin is an arginine-glycine-aspartic acid (RGD) cancer preventive peptide. The objective was to evaluate the potential of lunasin to induce apoptosis in human colon cancer cells and their oxaliplatin-resistant (OxR) variants, and its effect on the expression of human extracellular matrix and adhesion genes.
METHODS AND RESULTS: Various human colon cancer cell lines which underwent metastasis were evaluated in vitro using cell flow cytometry and fluorescence microscopy. Lunasin cytotoxicity to different colon cancer cells correlated with the expression of α(5) b(1) integrin, being most potent to KM12L4 cells (IC(50) = 13 μM). Lunasin arrested cell cycle at G2/M phase with concomitant increase in the expression of cyclin-dependent kinase inhibitors p21 and p27. Lunasin (5-25 μM) activated the apoptotic mitochondrial pathway as evidenced by changes in the expressions of Bcl-2, Bax, nuclear clusterin, cytochrome c and caspase-3 in KM12L4 and KM12L4-OxR. Lunasin increased the activity of initiator caspase-9 leading to the activation of caspase-3 and also modified the expression of human extracellular matrix and adhesion genes, downregulating integrin α(5), SELE, MMP10, integrin β(2) and COL6A1 by 5.01-, 6.53-, 7.71-, 8.19- and 10.10-fold, respectively, while upregulating COL12A1 by 11.61-fold.
CONCLUSION: Lunasin can be used in cases where resistance to chemotherapy developed.
7. Complementary Roles in Cancer Prevention: Protease Inhibitor Makes the Cancer Preventive Peptide Lunasin Bioavailable Chia-Chien Hsieh, Blanca Hernandez-Ledesma, Hyun Jin Jeong, Jae Ho Park, Ben O. de Lumen, PLoS ONE, (2010), 5 (1), 1-9.
Background: The lower incidence of breast cancer among Asian women compared with Western countries has been partly attributed to soy in the Asian diet, leading to efforts to identify the bioactive components that are responsible. Soy Bowman Birk Inhibitor Concentrate (BBIC) is a known cancer preventive agent now in human clinical trials. Methodology/Principal Findings: The objectives of this work are to establish the presence and delineate the in vitro activity of lunasin and BBI found in BBIC, and study their bioavailability after oral administration to mice and rats. We report that lunasin and BBI are the two main bioactive ingredients of BBIC based on inhibition of foci formation, lunasin being more efficacious than BBI on an equimolar basis. BBI and soy Kunitz Trypsin Inhibitor protect lunasin from in vitro digestion with pancreatin. Oral administration of 3H-labeled lunasin with lunasin-enriched soy results in 30% of the peptide reaching target tissues in an intact and bioactive form. In a xenograft model of nude mice transplanted with human breast cancer MDA-MB-231 cells, intraperitoneal injections of lunasin, at 20 mg/kg and 4 mg/kg body weight, decrease tumor incidence by 49% and 33%, respectively, compared with the vehicle-treated group. In contrast, injection with BBI at 20 mg/kg body weight shows no effect on tumor incidence. Tumor generation is significantly reduced with the two doses of lunasin, while BBI is ineffective. Lunasin inhibits cell proliferation and induces cell death in the breast tumor sections. Conclusions/Significance: We conclude that lunasin is actually the bioactive cancer preventive agent in BBIC, and BBI simply protects lunasin from digestion when soybean and other seed foods are eaten by humans.
8. Lunasin, with an arginine-glycine-aspartic acid motif, causes apoptosis to L1210 leukemia cells by activation of caspase-3
Elvira Gonzalez de Mejia, W. Wang, Vermont P. Dia, Molecular Nutrition & Food Research, (2010), 54 (3),
Lunasin is a novel chemopreventive peptide featuring a cell adhesion motif composed of arginine-glycine-aspartate (RGD) which has been associated to cytotoxicity to established cell lines. The objectives of this study were to determine the effect of lunasin on the viability of L1210 leukemia cells and to understand the underlying mechanisms involved. Pure lunasin and lunasin enriched soy flour (LES) caused cytotoxicity to L1210 leukemia cells with IC(50) of 14 and 16 microM (lunasin equivalent), respectively. Simulated gastrointestinal digestion showed that 25% of the original amount of lunasin survived 3 h of pepsin digestion and 3% of lunasin remained after sequential pepsin-pancreatin digestion for a total of 6 h. Cell cycle analysis showed that lunasin caused a dose-dependent G2 cell cycle arrest and apoptosis. Treatment of L1210 leukemia cells with 1 mg/mL of LES for 18 h led to an increase in the amount of apoptotic cells from 2 to 40%. Compared to untreated cells, treatment with 1 mg/mL LES showed a 6-fold increase on the expressions of caspases-8 and -9, and and a 12-fold increase on the expression of caspase-3. These results showed for the first time that lunasin, a naturally occurring peptide containing an RGD motif, caused apoptosis to L1210 leukemia cells through caspase-3 activation.
9. Lunasin promotes apoptosis in human colon cancer cells by mitochondrial pathway activation and induction of nuclear clusterin expression
Vermont P. Dia, Elvira Gonzalez de Mejia, The Cancer Letter, (2010), 295 (1), 44-53.
Lunasin is a naturally occurring peptide with arginine-glycine-aspartic acid motif associated to its reported biological activity. We aimed to determine the potential of lunasin from soybean to stimulate apoptosis in HT-29 colon cancer cells. Lunasin caused cytotoxicity to HT-29 cells and induced G2/M cell cycle arrest with simultaneous increased in p21 expression. Lunasin-induced apoptosis as evidenced by a twofold increase in the percentage of cells undergoing apoptosis, decreased Bcl-2:Bax ratio from 8.5 to 0.4, increased caspase-3 activity by 77% and increased expression of pro-apoptotic nuclear clusterin by five fold when compared to untreated cells. In conclusion, lunasin stimulated apoptosis in HT-29 cells by activating apoptotic mitochondrial pathways and inducing expression of the pro-apoptotic nuclear clusterin.
10. Recombinant expression of bioactive peptide lunasin in Escherichia coli
Chin-Feng Liu, Tzu-Ming Pan, Applied Microbiology and Biotechnology, (2010), 88 (1), 177-186.
Lunasin, a cancer-preventive peptide, was isolated from soybean, barley, and wheat. Previous studies showed that this 43-amino acid peptide has the ability to suppress chemical carcinogen-induced transformation in mammalian cells and skin carcinogenesis in mice. In this study, we attempted to use the Escherichia coli T7 expression system for expression of lunasin. The lunasin gene was synthesized by overlapping extension polymerase chain reaction and expressed in E. coli BL21(DE3) with the use of vector pET29a. The recombinant lunasin containing his-tag at the C-terminus was expressed in soluble form which could be purified by immobilized metal affinity chromatography. After 4 h, the expression level is above 4.73 mg of recombinant his-tagged lunasin/L of Luria-Bertani broth. It does not affect the bacterial growth and expression levels. This is the first study that successfully uses E. coli as a host to produce valuable bioactive lunasin. The result of in vitro bioassay showed that the purified recombinant lunasin can inhibit histone acetylation. Recombinant lunasin also inhibits the release of pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide production). Compared with other research methods on extraction or chemical synthesis to produce lunasin, our method is very efficient in saving time and cost. In the future, it could be applied in medicine and structure-function determination.
11. The role of nutraceutical proteins and peptides in apoptosis, angiogenesis, and metastasis of cancer cells
Elvira Gonzalez de Mejia, Vermont P. Dia, Cancer and Metastasis Reviews, (2010), 29 (3), 511-528.
The process of carcinogenesis is complex and not easy to eliminate. It includes the initial occurrence of genetic alterations which can lead to the inactivation of tumor-suppressor genes and further accumulation of genetic alterations during tumor progression. Looking for food and food components with biological properties, collectively called nutraceuticals, that can hinder such alterations and prevent the inactivation of tumor-suppressor genes is a very promising area for cancer prevention. Proteins and peptides are one group of nutraceuticals that show potential results in preventing the different stages of cancer including initiation, promotion, and progression. In this review, we summarized current knowledge on the use of nutraceutical proteins and peptides in cancer prevention and treatment. We focused on the role of plant protease inhibitors, lactoferrin and lactoferricin, shark cartilage, plant lectins, and lunasin in the apoptosis, angiogenesis, and metastasis of cancer cells. Also included are studies on bioavailability and clinical trials conducted on these promising proteins and peptides.
12. Soybean Peptide Lunasin Suppresses in Vitro and In Vivo 7,12-Dimethylbenz [a]anthracene-induced Tumorigenesis
Chia-Chien Hiseh, Blanca Hernández-Ledesma, Ben O. de Lumen, Journal of Food Science (2010), 75 (9), H311-H316.
Lunasin is a novel peptide identified in soybean and other seeds. This study evaluated the anti-tumorigenic effects of lunasin on 7,12-dimethylbenz(a)anthracene (DMBA) and 3-methylcholanthrene-treated (MCA) fibroblast NIH/3T3 cells. Lunasin significantly inhibited cell proliferation and cancerous foci formation in these 2 chemical carcinogens-treated cells. An in vivo SENCAR mouse model induced with DMBA was used to study the mammary cancer preventive properties of dietary lunasin contained in soy protein. Tumor incidence was 67% and 50%, and the tumor generation was 1.88 ± 0.48 and 1.17 ± 0.17, respectively, for the mice fed control diet and experimental diet obtained after AIN-93G supplementation with lunasin-enriched soy protein concentrate (containing 0.23% lunasin). However, no effects were observed in mice fed AIN-93G supplemented with soy protein concentrate (containing 0.15% lunasin). The data provided illustrate the anticancer potential of lunasin both in vitro and in vivo and supports the recommendation of soy protein as a dietary component that may aid in the prevention of mammary cancer.
13. Lunasin, a novel seed peptide, sensitizes human breast cancer MDA-MB-231 cells to aspirin-arrested cell cycle and induced apoptosis
Chia-Chien Hsieh, Blanca Hernández-Ledesma, Ben O. de Lumen, Chemico-Biological Interactions (2010), 186 (2), 127-134.
Breast cancer is one of the most common tumors in women of Western countries. The high aggressiveness and therapeutic resistance of estrogen-independent breast tumors have motivated the development of new strategies for prevention and/or treatment. Combinations of two or more chemopreventive agents are currently being used to achieve greater inhibitory effects on breast cancer cells. This study reveals that both aspirin and lunasin inhibit, in a dose-dependent manner, human estrogen-independent breast cancer MDA-MB-231 cell proliferation. These compounds arrest the cell cycle in the S- and G1-phases, respectively, acting synergistically to induce apoptosis. To begin elucidating the mechanism(s) of action of these compounds, different molecular targets involved in cell cycle control, apoptosis and signal transduction have been evaluated by real-time polymerase chain reaction (RT-PCR) array. The cell growth inhibitory effect of a lunasin/aspirin combination is achieved, at least partially, by modulating the expression of genes encoding G1 and S-phase regulatory proteins. Lunasin/aspirin therapy exerts its potent pro-apoptotic effect is at least partially achieved through modulating the extrinsic-apoptosis dependent pathway. Synergistic down-regulatory effects were observed for ERBB2, AKT1, PIK3R1, FOS and JUN signaling genes, whose amplification has been reported as being responsible for breast cancer cell growth and resistance to apoptosis. Therefore, our results suggest that a combination of these two compounds is a promising strategy to prevent/treat breast cancer.
14. Lunasin peptide purified from Solanum nigrum L. protects DNA from oxidative damage by suppressing the generation of hydroxyl radical via blocking fenton reaction
Jin Boo Jeong, Ben O. de Lumen, Hyung Jin Jeong, Cancer Letters (2010), 293 (1), 58-64.
Oxidative DNA damage is the most critical factor implicated in carcinogenesis and other disorders. However, the protective effects of lunasin against oxidative DNA damage have not yet reported. In this study, we report here the protective effect of lunasin purified from Solanum nigrum L. against oxidative DNA. Lunasin protected DNA from the oxidative damage induced by Fe2+ ion and hydroxyl radical. To better understand the mechanism for the protective effect of lunasin against DNA damage, the abilities to chelate Fe2+, scavenge the generated hydroxyl radical and block the generation of hydroxyl radical were evaluated. Although it did not scavenge generated hydroxyl radical, lunasin blocked the generation of hydroxyl radical by chelating Fe2+ ion. We conclude that lunasin protects DNA from oxidation by blocking fenton reaction between Fe2+ and H2O2 by chelating Fe2+ and that consumption of lunasin may play an important role in the chemoprevention for the oxidative carcinogenesis.
15. Lunasin and lunasin-like peptides inhibit inflammation through suppression
of NF-kB pathway in the macrophage
Elvira Gonzalez de Mejia, Vermont P. Dia, Peptides, (2009), 30, 2388-2398.
Inflammation is part of the host defense mechanism against harmful matters and injury; however, aberrant inflammation is associated to the development of chronic diseases such as cancer. Lunasin is a novel peptide that demonstrates potential anticancer activity against mammalian cancer cell lines and may play a role in inflammation. The objective of this study was to determine the mechanism of action by which lunasin and lunasin-like peptides exert their anti-inflammatory properties using RAW 264.7 macrophage cell line as an in vitro model. We purified three peptides (5, 8, and 14 kDa) from defatted soybean flour with a positive immunoreactivity towards lunasin mouse monoclonal antibody. Treatment with these peptides (10–50 mM) resulted in the inhibition of pro-inflammatory markers in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. The 5 kDa peptide inhibited most potently pro-inflammatory markers including interleukin-6 production (IC50 = 2 mM), interleukin-1b production (IC50 = 13 mM), nuclear factor-kappa B (NF-kB) transactivation (IC50 = 21 mM), cyclooxygenase-2 expression (IC50 = 25 mM), nitric oxide production (IC50 = 28 mM), inducible nitric oxide synthase expression (IC50 = 37 mM), prostaglandin E2 production (IC50 = 41 mM), p65 nuclear translocation (IC50 = 48 mM) and p50 nuclear translocation (IC50 = 77 mM). In conclusion, lunasin and lunasin-like peptides purified from defatted soybean flour inhibited inflammation in LPS-induced RAW 264.7 macrophage by suppressing NF-kB pathway.
16. Lunasin and Bowman-Birk protease inhibitor (BBI) in US commercial soy foods
Blanca Hernández-Ledesma, Chia-Chien Hsieh, Ben O. de Lumen, Food Chemistry, (2009), 115, 574-580.
The inverse association between the intake of soybean foods and cancer incidence and mortality rates supported by published literature has led to studies on identifying bioactive components. The cancer preventive properties of the soybean peptides lunasin and Bowman-Birk protease inhibitor (BBI) have been demonstrated by in vitro and in vivo assays. Both peptides were present in most commercially available soy products, in varying concentrations, depending mainly on the soybean variety and the manufacturing process. Lunasin and BBI were absent in the fermentation products natto and miso, suggesting that fermentation destroys both peptides. To study the bioavailability of lunasin and BBI, three soy milk samples with different concentrations of these peptides
were subjected to an enzymatic hydrolysis process simulating physiological digestion. The results confirm the important role BBI plays in the protection of lunasin from digestion by pepsin and pancreatin.
17. Isolation, purification and characterisation of lunasin from defatted soybean
flour and in vitro evaluation of its anti-inflammatory activity
V.P. Dia , W. Wang, V.L. Oh, B.O. de Lumen, E. Gonzalez de Mejia
Food Chemistry, (2009), 114, 108–115.
Lunasin was also found to inhibit COX-2/PGE2 and iNOS/NO pathways. This newly discovered property of lunasin might contribute to the suppression of inflammation in vivo.
18. Presence of Lunasin in Plasma of Men after Soy Protein Consumption
Vermont P. Dia, Sofia Torres, Ben O. de Lumen, John W. Erdman, JR. Elvira de Mejia, J. Agric. Food Chem., (2009), 57, 1260–1266.
Liquid chromatography-tandem mass spectrometry analysis showed the presence of amino acid sequences from lunasin in plasma samples after soy intake for 30 min and 1 hour. No peptides from lunasin were present in plasma samples without soy intake. The results of this study suggest that lunasin is bioavailable in humans, an important requirement for its anticancer potential.
19. Antioxidant and anti-inflammatory properties of cancer preventive peptide lunasin in RAW 264.7 macrophages
Chia-Chien Hsieh, Blanca Hernández-Ledesma, Ben O. de Lumen
Biochemical and Biophysical Research Communications (2009), 390 (3), 803-808.
Oxidative stress and inflammation are two of the most critical factors implicated in carcinogenesis and other degenerative disorders. We have investigated how lunasin, a known anti-cancer seed peptide, affect these factors. This peptide inhibits linoleic acid oxidation and acts as 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenger. Furthermore, using LPS-stimulated RAW 264.7 macrophages, we have demonstrated that lunasin reduces, in a significant dose-dependent manner, the production of reactive oxygen species (ROS) by LPS-induced macrophages. Lunasin also inhibits the release of pro-inflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukine-6 [IL-6]). On the basis of these potent antioxidant and anti-inflammatory properties, we propose lunasin not only as a cancer preventive and therapeutic agent but also as an agent against other inflammatory-related disorders.
20. Lunasin, a novel seed peptide for cancer prevention
Blanca Hernandez-Ledesma, Chia-Chien Hsieh, Ben O. de Lumen, Peptides, (2009), 30, 426-430 and Perspectives in Medicinal Chemistry (2008) 2, 75-80.
Carcinogenesis is a multistage process derived from a combination of multiple heritable and environmental factors. It has been reported that populations consuming high levels of soybeans products have both lower cancer incidence and mortality rates in the western countries. Lunasin is a novel and promising peptide initially discovered in soy and now found in wheat, barley and other seeds. Its cancer-preventative efficacy has been shown in mammalian cells which were induced by chemical carcinogens and viral oncogenes. Moreover, this peptide has been found to prevent skin cance4r in a mouse cancer model induced by chemical carcinogens. Its bioavailability after oral administration makes it a perfect candidate as a chemopreventative agent. The purpose of this article is to review the discovery of this seed peptide and the most recent evidence on its possible benefits as an anticancer agent.
21. Analysis of Soybean Protein- Derived Peptides and the Effect of Cultivar, Environmental Conditions, and Processing on Lunasin Concentration in Soybean and Soy Products
Wenyi Wang, Vermont P. Dia, Miguel Vasconez, Randall L. Nelson and Elvira Gonzalez de Mejia, Journal of the Association of Official Analytical Chemists International, (2008), 91 (4), 936-946.
Lunasin concentration was affected by cultivar-temperature, cultivar-soil moisture and cultivar-temperature-soil-moisture interactions. The variation on lunasin concentration suggests that its content can be improved by breeding, and by optimization of growing conditions. In summary, bioactive peptides can be accurately identified and quantified using different techniques and conditions. In addition, lunasin concentration in soybean depends mainly on cultivar and to some extent on environmental factors particularly temperature. Lunasin concentration in soy products was also affected by processing conditions.
22. Lunasin: A Novel Cancer Preventive Seed Peptide that Modifies Chromatin
Ben O. de Lumen, Journal of AOAC Int. (2008), 91 (4), 932-935.
Lunasin is a novel cancer preventive peptide whose efficacy against chemical carcinogens and oncogenes has been demonstrated in mammalian cells and a skin cancer mouse model. In contrast, constitutive expression of the lunasin gene in mammalian cells leads to arrest of cell division and cell death. Isolated and characterized in soy, lunasin peptide is also documented in barley and wheat and is predicted to be present in many more seeds because of its possible role in seed development. Initial studies show that lunasin is bioavailable in mice when orally ingested. Lunasin internalizes into mammalian cells within minutes of exogenous application, and localizes in the nucleus after 18 h. It inhibits acetylation of core histones in mammalian cells but does not affect the growth rate of normal and established cancer cell lines. An epigenetic mechanism of action is proposed whereby lunasin selectively kills cells being transformed or newly transformed cells by binding to deacetylated core histones exposed by the transformation event, disrupting the dynamics of histone acetylation–deacetylation.
23. Bowman-Birk Inhibitor and Genistein among Soy Compounds That Synergistically Inhibit Nitric Oxide and Prostaglandin E2 Pathways in Lipopolysaccharide-Induced Macrophages
Vermont P. Dia, Mark A. Berhow, and Elvira Gonzalez de Mejia
J. Agric. Food Chem., (2008) 56 (24), 11707-11717.
Inflammation has an important role in the development of chronic diseases. In this study, we evaluated the anti-inflammatory properties of eight soybean bioactive compounds by measuring their ability to inhibit cyclooxygenase-2/prostaglandin E2 (PGE2) and inducible nitric oxide synthase (iNOS)/nitric oxide (NO) inflammatory pathways. Saponin A group glycosides showed inhibition of the iNOS/NO pathway only, while pepsin-pancreatin glycinin hydrolysates (lunasin) enhanced induction and production of the four inflammatory responses. For the first time, synergistic interactions were observed between BBI and genistein inhibiting NO (92.7%) and PGE2 (95.6%) production. These findings demonstrated that some SBCs possess anti-inflammatory properties and therefore are important in modulating mammalian inflammation pathways which may lead to inhibition of some types of chronic disease. Furthermore, through their interaction they can modulate the inflammatory process.
24. Bioactive Peptides in Amaranth (Amaranthus hypochondriacus) Seed
C. Silva-Sanchez, A. P. Barba De LaRosa, M. F. Leon-Galvan, B. O. de Lumen, A. DeLeon Rodriguez, And E. Gonzalez de Mejia, J. Agric. Food Chem. (2008), 56, 1233–1240.
This is the first study that reports the presence of a lunasin-like peptide and other potentially bioactive peptides in amaranth protein fractions. Lunasin was also identified in albumin, prolamin and globulin amaranth protein fractions and even in popped amaranth seeds. Western blot analysis revealed a band at 18.5 kDa, and MALDITOF analysis showed that this peptide matched more than 60% of the soybean lunasin peptide sequence.
25. Role of Dietary Soy Protein in Obesity- a Review
Manuel T. Velasquez and Sam J. Bhathena
International Journal of Medical Sciences, (2007), 4 (2), 72-82.
Soy protein is an important component of soybeans and provides an abundant source of dietary protein. Among the dietary proteins, soy protein is considered a complete protein in that it contains ample amounts of all the essential amino acids plus several other macronutrients with a nutritional value roughly equivalent to that of animal protein of high biological value. Several potential mechanisms whereby soy protein may improve insulin resistance and lower body fat and blood lipids are discussed and include a wide spectrum of biochemical and molecular activities that favorably affect fatty acid metabolism and cholesterol homeostasis. The biologic actions of certain constituents of soy protein, particularly conglycinin, soya saponins, phospholipids, and isoflavones, that relate to obesity are also discussed.
26. In Vitro Digestibility of the Cancer-Preventive Soy Peptides Lunasin and BBI
Jae Ho Park, Hyung Jin Jeong, Ben O. de Lumen
J. Agric. Food Chem. (2007), 55 (26), 10703-10706.
Lunasin and BBI (Bowman Birk protease inhibitor) are bioactive soy peptides that have been shown to be effective suppressors of carcinogenesis in in vitro and in vivo model systems. Since they are subject to digestion in the gastrointestinal tract, we investigated here the stabilities of lunasin and BBI to digestion in vitro by simulated intestinal fluid (SIF) and simulated gastric fluid (SGF). Samples containing lunasin and BBI of varying purities were subjected to in vitro digestion by SIF and SGF at different times and analyzed by Western blot. While the pure BBI reaction is stable after SIF and SGF digestions, the purified lunasin from soybean and synthetic lunasin are easily digested after 2 min in both in vitro digestions. In contrast, lunasin from soy protein containing BBI is comparatively stable after SIF and SGF digestions. Both lunasin and BBI are able to internalize into the cell and localize in the nucleus even after digestion, suggesting that some of the peptides are intact and bioactive. These data suggest that BBI plays a role in protecting lunasin from digestion when soy protein is consumed orally. The role of other soy protease inhibitors such as Kunitz Trypsin Inhibitor (KTI) cannot be excluded from these experiments.
27. Cancer-Preventive Peptide Lunasin from Solanum nigrum L. Inhibits Acetylation of Core Histones H3 and H4 and Phosphorylation of Retinoblastoma Protein (Rb)
Jin Boo Jeong, Hyung Jin Jeong, Jae Ho Park, Sun Hee Lee, Jeong Rak Lee, Hee Kyeong Lee, Gyu Young Chung, Jeong Doo Cho, and Ben O. de Lumen, J. Agric. Food Chem., (2007), 55 (26), 10707-10713.
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.
28. Inhibition of Core Histone Acetylation by the Cancer Preventive Peptide Lunasin
Hyung Jin Jeong, Jin Boo Jeong, Dae Seop Kim, Ben O. de Lumen
J. Agric. Food Chem. (2007), 55 (3), 632-637.
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.
29. The Potential Allergenicity of Two 2S Albumins from Soybean (Glycine max) : A Protein Microarray Approach
Jing Lin, Peter R. Shewry, David B. Archer, Kirsten Beyer, Bodo Niggemann, Helmut Haas, Philip Wilson, Marcos J.C. Alcocer, International Archives of Allergy and Immunology, (2006), 141, 91–102.
The 2S albumins are a group of storage proteins that occur widely in seeds of dicotyledonous plants. The widespread distribution and stability to digestion of allergenic 2S albumins raise the question of why some members of this family present in important food sources, such as soybean, are not regarded as major allergens. None of the patients was found to have IgE specific to soybean 2S albumins. The results from microarray, micro-ELISA and UniCAP system suggested that the 2S albumins from soybean are not major allergens within the patient population analyzed.
30. Contents and Bioactivities of Lunasin, Bowman-Birk Inhibitor, and Isoflavones in Soybean Seed
Jae Ho Park, Hyung Jin Jeong, Ben O. de Lumen
J. Agric. Food Chem. (2005), 53 (20), 7686-7690.
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.
31. Lunasin: A Cancer-Preventive Soy Peptide
Ben O. de Lumen.
Nutrition Reviews (2005), 63 (1), 16-21.
Lunasin is a novel, cancer-preventive peptide whose efficacy against chemical carcinogens and oncogenes has been demonstrated in mammalian cells and in a skin cancer mouse model. Isolated and characterized in soy, lunasin peptide is also documented in barley. Lunasin is found in all of the genotypes analyzed from the US soy germ plasm collection and in commercially available soy proteins. Pilot studies show that lunasin is bioavailable in mice and rats when orally ingested, opening the way for dietary administration in cancer prevention studies. Lunasin internalizes into mammalian cells within minutes of exogenous application, and localizes in the nucleus after 18 hours. It inhibits acetylation of core histones in mammalian cells. In spite of its cancer-preventive properties, lunasin does not affect the growth rate of normal and established cancer cell lines. An epigenetic mechanism of action is proposed whereby lunasin selectively kills cells being transformed or newly transformed by binding to deacetylated core histones exposed by the transformation event, disrupting the dynamics of histone acetylation-deacetylation and leading to cell death.
32. Dynamics of Keratinocytes in Vivo using 2H20 Labeling: A Sensitive Marker of Epidermal Proliferation State
Elaine A. Hsieh, Christine M. Chai, Ben O. de Lumen, Richard A. Neese, Marc K. Hellerstein, Journal of Investigative Dermatology, (2004), 123 (3), 530-536.
A heavy water (2H2O) labeling method recently developed to measure cell proliferation in vivo is applied here to the measurement of murine epidermal cell turnover and to investigate conditions in which keratinocyte proliferation is either inhibited or stimulated. The technique is based on incorporation of 2H2O into the deoxyribose moiety of deoxyribonucleotides in dividing cells. Label incorporation and die-away studies in cells isolated from C57BL/6J mouse epidermis revealed the replacement rate to be 34%-44% per wk (half-life of 1.6-2 wk). The kinetics provided evidence of a non-proliferative subpopulation of cells (10%-15% of the total) within the epidermis. Topical administration of 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate for 3 wk increased epidermal cell proliferation by 55% in SENCAR mice. Topical addition of lunasin, an anti-mitotic agent from soy, decreased epidermal cell proliferation modestly though significantly (16% given alone, 9% given with carcinogens). Caloric restriction (by 33% of energy intake) for 4 wk decreased the epidermal cell proliferation rate by 45% in C57BL/6J mice. In summary, epidermal cell proliferation can be measured in vivo using 2H20 labeling in normal, hyper- and hypo-proliferative conditions. Potential applications of this inherently safe method in humans might include studies of psoriasis, wound healing, chemopreventive agents, and caloric intake.
33. Lunasin Concentration in Different Soybean Genotypes, Commercial Soy
Protein, and Isoflavone Products
Elvira Gonzalez de Mejia, Miguel Vásconez, Ben O. de Lumen, and Randall Nelson
J. Agric. Food Chem., (2004), 52 (19), 5882 -5887.
Lunasin concentrations in the tested materials range from 0.10 to 1.33 g/100 g flour. Differences that exceeded 100% have been observed among accessions of similar maturity that were grown in the same environment, indicating that genetic differences in soybeans exist for Lunasin.
34. The Anticarcinogenic Potential of Soybean Lectin and Lunasin- Special Article
Elvira Gonzalez de Mejia, Ph.D., Traliece Bradford, B.S., and Clare Hasler, Ph.D.
International Life Sciences Institute, Nutrition Reviews, (2003), 61 (7), 239–246.
Soybeans contain a variety of anticarcinogenic phytochemicals. Recently, there has been increased interest in the potential health benefits of bioactive polypeptides and proteins from soybeans, including lunasin and lectins. Lunasin is a polypeptide that arrests cell division and induces apoptosis in malignant cells. Additional research, including clinical trials, should continue to examine and elucidate the therapeutic effects, nutritional benefits, and toxic consequences of commonly ingested soybean lectins and lunasin.
35. Lunasin Suppresses E1A-Mediated Transformation of Mammalian Cells But Does Not Inhibit Growth of Immortalized and Established Cancer Cell Lines
Yi Lam, Alfredo Galvez and Ben O. de Lumen, Nutrition and Cancer, (2003), 47 (1), 88-94.
Within 18 hours exogenous application, Lunasin internalizes into the cell and localizes in the nucleus. In contrast to its inhibitory effects on cell transformation, lunasin has no effect on growth of immortalized (non-tumorgenic) and established cancer cells. This is the first report that lunasin suppresses transformation of mammalian cells induced by an oncogene (E1A) in addition to chemical carcinogens.
36. Characterization of Lunasin Isolated from Soybean
Hyung Jin Jeong, Jae H. Park, Yi Lam, Ben O. de Lumen
J. Agric. Food Chem. (2003), 51 (27), 7901-7906.
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.
37. Barley Lunasin Suppresses ras-Induced Colony Formation and Inhibits Core Histone Acetylation in Mammalian Cells
Hyung J. Jeong, Yi Lam, and Ben O. de Lumen, J. Agric. Food Chem., (2002), 50, 5903-5908.
Lunasin is a novel peptide originally identified in soybean that suppresses chemical carcinogen induced transformation in mammalian cells and skin carcinogenesis in mice. We report here the isolation, purification, and biological assay of lunasin from barley, a newly found rich source of the peptide. 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.
38. Identification and bioavailability of a chromatin-binding peptide (Lunasin) from Korean Soybean
Jae Ho Park, Soon Young Kim, Ben O. de Lumen, Kon Joo Lee and Hyung Jin Jeong, Journal of Plant Biology, (2002), 45 (2), 96-101.
Lunasin is a novel peptide with great potential as a nontoxic chemopreventive drug. This compound might account for part of the anticancer effects reported from studies with soybean(Glycine max). We studied its isolation, purification and biological assay, and observed that both its band from soybean and one from synthetic lunasin were <5 kD in their molecular weights. Among all the crop varieties tested, only the soybean produced a lunasin band on our western blot. Levels of this peptide ranged from 0.045 mg per gram of seed for the Hanbatkong cultivar to 0.156 mg per gram seed for Poolunkong’. The effect on colony formation by lunasin from different soybean extracts was significantly higher than for either the positive control or the synthetic lunasin. When lunasin was present in those natural extracts, histone acetylation decreased 100% compared with cells that were treated with Na-butyrate.
39. Chemopreventive Property of a Soybean Peptide (Lunasin) That Binds to Deacetylated Histones and Inhibits Acetylation
Alfredo F. Galvez, Na Chen, Janet Macasieb, and Ben O. de Lumen
Cancer Research, (2001), 61, 7473–7478.
This study shows that exogenous application of the lunasin peptide inhibits chemical carcinogen-induced transformation of murine fibroblast cells to cancerous foci. These results suggest a mechanism whereby lunasin selectively induces apoptosis, mostly in cells undergoing transformation, by preventing histone acetylation. In support of this, lunasin selectively induces apoptosis in E1A-transfected cells but not in non-transformed cells. Finally, in the SENCAR mouse skin cancer model, dermal application of lunasin (250 µg/week) reduces skin tumor incidence by ~70%, decreases tumor yield/mouse, and delays the appearance of tumors by 2 weeks relative to the positive control. These results point to the role of lunasin as a new chemopreventive agent that functions possibly via a chromatin modification mechanism.
40. A soybean cDNA encoding a chromatin binding peptide inhibits mitosis of mammalian cells
Alfredo F. Galvez and Benito O. de Lumen, Nature America, (1999), 17, 495-500.
A soybean cDNA encoding the small subunit peptide (called Lunasin) of a cotyledon-specific 2S albumin (Gm2S-1) is thought to play a role in arresting mitosis during the DNA endoreduplication and cell expansion phase of seed development. Observations in this study suggest that Lunasin binds to the chromatin, leading to disruption of kinetochore formation and inhibition of mitosis.
41. Epigenetic Regulation of HMG-CoA Reductase and LDL Receptor Expression By The Soy Peptide Lunasin Provides Mechanism for Lowering Cholesterol
Alfredo F. Galvez, Fu Chunjiang, Jim Porter, Monty Kerley, (presented abstract).
The American Heart Association recently reported that soy protein lowers LDL cholesterol by only 3%, contrary to the FDA health claim on soy protein. We now report the discovery of a chromatin-binding peptide from soy that could very well be the active factor responsible for the LDL cholesterol-lowering effect attributed to soy proteins and help clarify clinical results summarized in the recent AHA report. Lunasin is a recently discovered bioactive soy component with a novel chromatin binding property. Lunasin has been shown to significantly reduce the acetylation of histone H3 by the histone acetylase enzyme, PCAF (p300/CBP-associated factor) but not by p300 or HAT1. Transcriptional regulation of HMG CoA reductase and LDL receptor genes by Sterol Regulatory Element Binding Proteins (SREBP) in cholesterol-free media requires the selective recruitment of different co-regulatory factors, CREB/CBP for HMG-CoA reductase and SP1 for LDL receptor. CREB-binding protein (CBP) interacts with the activation domain of SREBP and has been shown to associate with PCAF to specifically acetylate histone H3. The acetylation of histone H3 by PCAF is required for SREBP-controlled transcriptional activation of HMG-CoA reductase and by inhibiting H3 acetylation, lunasin can potentially reduce its expression. Cell culture of HepG2 liver cells shows that treatment with lunasin can indeed significantly reduce HMG-CoA reductase expression by 50% in cholesterol-free media. In contrast, LDL receptor expression has increased by 60%, which can be explained in part by the coordinate increase in expression of its co-transcriptional activator, Sp1. Also, the inhibition of HMG-CoA reductase expression by lunasin lowers intracellular cholesterol levels that keeps SREBP activated, contributing to the upregulation of LDL receptor expression. Results from RT-PCR experiments show the corresponding reduction of HMG-CoA reductase and the increase of LDL receptor mRNA transcript levels in lunasin-treated, cholesterol-free media, which has also been validated by real-time quantitative RT-PCR. Hence, these studies show that lunasin has the potential to reduce LDL and total cholesterol levels by directly inhibiting gene expression of HMG-CoA reductase, which reduces cholesterol biosynthesis, and by increasing LDL receptor expression, which enhances clearance of plasma LDL cholesterol. The presence of the lunasin peptide in soy protein preparations provides a plausible mechanism of action to explain the cholesterol-lowering effect attributed to soy protein and paves the way for optimizing soy protein ingredients to maximize its heart-healthy benefits.