WO2008034117A2

WO2008034117A2 - Methods for using soy peptides to inhibit h3 acetylation, reduce expression of hmg-coa reductase and increase ldl receptor and sp1 expression in a mammal

Info

Publication number: WO2008034117A2
Application number: PCT/US2007/078585
Authority: WO
Inventors: Alfredo Flores Galvez
Original assignee: Alfredo Flores Galvez
Priority date: 2006-09-16
Filing date: 2007-09-15
Publication date: 2008-03-20
Also published as: WO2008034117A3

Abstract

Controlled studies demonstrate that methods using soy related peptides inhibit H3 acetylation, reduce expression of HMG-CoA reductase and increase LDL receptor and Sp1 expression in mammals. The present disclosure is generally directed to using lunasin peptides and/or lunasin peptide derivatives to 1) inhibit H3 acetylation, 2) reduce expression of HMG-CoA reductase, 3) increase LDL receptor expression or 4) increase Sp1 expression in a mammal. In at least one exemplary embodiment of the present disclosure, an effective amount of lunasin peptides or lunasin peptide derivatives and one or more enzyme inhibitors is provided to a mammal to 1) inhibit H3 acetylation, 2) reduce expression of HMG-CoA reductase, 3) increase LDL receptor expression or 4) increase Sp1 expression in a mammal.

Description

METHODS FOR USING SOY PEPTIDES TO INHIBIT H3 ACETYLATION,

REDUCE EXPRESSION OF HMG-COA REDUCTASE AND INCREASE

LDL RECEPTOR AND SP1 EXPRESSION IN A MAMMAL BY

ALFREDO FLORES GALVEZ

RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Ser. No. 1 1/532,528 filed September 16, 2006. This application also claims the benefit of priority to U.S. Provisional Application Number , filed September 16, 2006. This application also claims the benefit of priority to International Application Number , filed

September 15, 2007. All of the above listed applications are hereby incorporated by reference herein in their entireties.

BACKGROUND [0002] TECHNICAL FIELD

[0003] This disclosure relates generally to a class of peptides that provide mammals with a variety of health related benefits. More specifically, the present disclosure related to using soy peptides to inhibit H3 acetylation, reduce expression of HMG-CoA reductase and increase LDL receptor and Sp1 expression in a mammal. [0004] BACKGROUND ART

[0005] Being able to control or manipulate certain important biological processes provides numerous benefits to researchers and individuals alike. The ability to effect expression of important receptors, enzymes and activators allows researchers to better understand complex biological mechanisms and create novel and beneficial therapies. For example, H3 acetylation, expression of HMG-CoA reductase and LDL receptor and Sp1 expression in mammals pays a significant role in various health related factors, including but not limited to total and cholesterol levels and cancer prevention. Accordingly, manipulation and control of these biological mechanisms or factors would provide numerous health related benefits and allow researches with new avenues to develop new therapies. Unfortunately, presently there are no known effective methods of safely inhibiting H3 acetylation, reducing expression of HMG-CoA reductase and increasing LDL receptor and Sp1 expression in a mammal. The ability to influence these and other biological factors would be very beneficial to the fields of science and medicine. Accordingly, there exists a need for improved methods of inhibiting H3 acetylation, reducing expression of HMG-CoA reductase and increasing LDL receptor and Sp1 expression in a mammal. The present invention provides these and other related benefits.

DISCLOSURE OF THE INVENTION [0006] The present invention relates generally to a class of peptides that provide mammals with a variety of health related benefits. More specifically, the present invention involves using soy peptides to inhibit H3 acetylation, reduce expression of HMG-CoA reductase and increase LDL receptor and Sp1 expression in a mammal.

[0007] In at least one exemplary embodiment of the present invention, a method of inhibiting PCAF from acetylating H3 in a mammal is provided. The method includes providing an effective amount of lunasin peptides to a mammal to inhibit H3 acetylation in the mammal.

[0008] In at least one other exemplary embodiment of the present invention, a method of reducing expression of HMG-CoA reductase in a mammal is provided. The method includes providing an effective amount of lunasin peptides to a mammal to reduce expression of HMG-CoA reductase in the mammal.

[0009] In at least one other exemplary embodiment of the present invention, a method of increasing LDL receptor expression in a mammal is provided. The method includes providing an effective amount of lunasin peptides to a mammal to increase LDL receptor expression in the mammal.

[0010] In at least one other exemplary embodiment of the present invention, a method of increasing Sp1 transcriptional activator expression in a mammal is provided. The method includes providing an effective amount of lunasin peptides to a mammal to increase Sp1 transcriptional activator expression in the mammal. [0011] In one aspect of at least one embodiment of the present invention, the effective amount of lunasin peptides that inhibit H3 acetylation, reduce expression of HMG-CoA reductase, increase LDL receptor expression or increases Sp1 transcriptional activator expression in a mammal is 25 to 100 mg daily.

[0012] In another aspect of at least one embodiment of the present invention, the lunasin peptides include lunasin peptides or lunasin peptide derivatives. [0013] In yet another aspect of at least one embodiment of the present invention, the lunasin peptides are obtained from, soy, seed bearing plants other than soy, using recombinant DNA techniques and synthetic polypeptide production or any combination thereof.

[0014] In yet another aspect of at least one embodiment of the present invention, the method includes providing an effective amount of one or more protease enzyme inhibitors to the lunasin peptides.

BRIEF DESCRIPTION OF DRAWINGS

[0015] The above-mentioned features and objects of the present disclosure will become more apparent with reference to the following description taken in conjunction with the accompanying drawings wherein like reference numerals denote like elements and in which:

[0016] Figure 1 shows the 2S albumin protein encoded by Gm2S 1 cDNA (SEQ ID NO 2). Arrows indicate endoproteolytic sites that give rise to small subunit (lunasin) (SEQ ID NO 2) and the large subunit (methionine rich protein). Important regions in both subunits are indicated.

[0017] Figure 2 is a photograph of a Western blot analysis (top) and a table (below) showing densitometer values indicating the relative levels of expression of HMG-CoA reductase in HepG2 cells that were (CFM+LS (24)) or were not (CFM) treated with lunasin for 24 hours prior to incubation in cholesterol free media (CFM) for 24 hours to activate sterol regulatory element binding proteins (SREBP.) After incubations, total protein was extracted and 10 ug protein was loaded onto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane, and immunostained with primary antibodies raised against HMG-CoA reductase and actin (to show equal loading of proteins.) Spot densitometer values represent mean and standard deviation of data from three separate experiments. [0018] Figure 3 is a photograph of a Western blot analysis (top) and a table (below) showing densitometer values indicating the relative levels of expression of LDL receptor in HepG2 cells that were (CFM +LS(24)) or were not (CFM) treated with lunasin for 24 hours prior to incubation in cholesterol free media (CFM) for 24 hours to activate SREBP. After incubations, total protein was extracted and 10 ug protein was loaded onto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane, and immunostained with primary antibodies raised against LDL-receptor and actin (to show equal loading of proteins.) Spot densitometer values represent mean and standard deviation of data from three separate experiments. [0019] Figure 4 is a photograph of a Western blot analysis (top) and a table (below) showing densitometer values indicating the relative levels of expression of Sp1 in HepG2 cells that were grown from confluence in growth media for 24 hours before growth media was replaced with fresh growth media (Media), media with lunasin (Media + LS) or cholesterol free media with lunasin (CFM +LS) or without lunasin (CFM). Samples were then incubated for 24 or 48 hours as indicated. After incubations, total protein was extracted and 10 ug protein was loaded onto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane, and immunostained with primary antibodies raised against Sp1 and actin (to show equal loading of proteins.) Spot densitometer values represent data from one experiment. [0020] Figure 5 shows the western blots from experiments on PCAF reaction products demonstrating that lunasin caused a dramatic reduction in histone H3 acetylation. Acid extracted protein from untreated (untrt) HeLa cells was used as template in histone acetylase reactions using HAT enzyme, PCAF, in the presence or absence of 10 uM lunasin. Reaction products were immunoblotted and stained with antibodies against diacetylated histone H3. Untrt (-) is the histone template control, NaB (+) correspond to acid extracted histones from NaButyrate treated HeLa cells (positive control). Boxed signal indicates significant decrease in H3 acetylation upon addition of 10 uM lunasin compared with no lunasin application. Numbers in parenthesis indicate densitometer readings relative to the untreated control (set as 1 ) in PCAF HAT reaction products.

[0021] Figure 6 shows the western blots from experiments on PCAF HAT reaction products demonstrating that lunasin caused a dramatic reduction in histone H3 acetylation. Acid extracted histones isolated from untreated (untrt) HeLa cells were used in PCAF HAT reactions, immunoblotted and stained with antibodies to H3 Ac-Lys9 and H3 Ac-Lys14. Untrt (-) is the histone template control, NaB (+) correspond to acid extracted histones from NaButyrate treated HeLa cells (posititve control), + Lun correspond to 10 uM lunasin treated histone template and - Lun correspond to non- lunasin treated. Boxed signal indicate decreased H3 Lys 14 acetylation by PCAF acetylase enzyme in the presence of lunasin. Numbers in parenthesis indicate densitometer readings relative to the signal level of lunasin/lunasin treatment (set as 1 ) in immunoblots stained with Ac-Lys14 H3.

DETAILED DESCRIPTION [0022] Lunasin is a recently discovered bioactive component in soy with a novel chromatin-binding property and epigenetic effects on gene expression (1 , 2). The lunasin soy peptide is heat stable, water soluble and found in significant amounts in select soy protein preparations (3). Studies show that it can get inside mammalian epithelial cells through its RGD cell adhesion motif, bind preferentially to deacetylated histones and inhibit histone H3 and H4 acetylation (4). There is growing evidence that cellular transformation, responses to hormones and dietary and environmental effects involve epigenetic changes in gene expression, which are modulated by the reversible processes of DNA methylation-demethylation and histone acetylation-deacetylation (5, 6). Lunasin is the first natural substance to be identified as a histone acetylase inhibitor, although it does not directly affect the histone acetylase enzyme. It inhibits H3 and H4 acetylation by binding to specific deacetylated lysine residues in the N-terminal tail of histones H3 and H4, making them unavailable as substrates for histone acetylation. The elucidation of the mechanism of action makes lunasin an important molecule for research studies to understand the emerging role of epigenetics and chromatin modifications in important biological processes.

[0023] The study on the effect of lunasin on prostate carcinogenesis at the University of California at Davis revealed the effects of lunasin on histone H4 modifications and the up regulation of chemopreventive genes, (7). However, until now, the specific effect of lunasin binding to deacetylated H3 N-terminal tail and the inhibition of H3 histone acetylation in biological systems had not yet been investigated. To determine the specific biological effect of lunasin binding to deacetylated histone H3 and inhibition of acetylation, the induction of genes involved in cholesterol biosynthesis by the sterol regulatory element binding proteins (SREBP) was chosen as a biological model. This biological model was chosen because activation of SREBPs by sterol depletion results in the increased acetylation of histone H3 but not histone H4, by the histone acetylase enzyme PCAF, in chromatin proximal to the promoters of HMG-CoA reductase and the LDL receptor genes (8) and SREBP activation results in the increased recruitment of co-regulatory factors, CREB to the promoter of HMG-CoA reductase gene, and Sp1 to the promoter of LDL receptor gene (8).

[0024] Our studies on in vitro histone acetylase (HAT) assays show that lunasin significantly inhibits histone H3 acetylation (specifically lysine 14 in H3 N-terminal tail) by the histone acetylase enzyme, PCAF. Cell culture experiments using HepG2 liver cells show that synthetic lunasin can significantly reduce HMG-CoA reductase expression and increase LDL receptor gene expression in cholesterol-free media similar to the effects of statin (cholesterol-lowering) drugs. Our studies have also shown that the increase in LDL receptor expression coincides with the increase in Sp1 expression in cholesterol-free media. Based on these studies, a molecular mechanism of action is proposed wherein synthetic lunasin reduces total and LDL cholesterol levels by binding to deactylated histone H3 and inhibiting histone H3 acetylation by PCAF (through its association with the CREB-binding protein), thereby reducing SREBP activation of the HMG-CoA reductase gene resulting in lower endogenous cholesterol biosynthesis, and by increasing the expression of the Sp1 co-activator in sterol-free media and upon SREBP activation, an increased amount of membrane bound LDL receptors is expressed leading to significant reduction of plasma LDL cholesterol levels (9).

[0025] Our data described and shown below demonstrates that lunasin (a.k.a. lunastantin) is the bioactive agent from soy responsible for inhibiting H3 acetylation, reducing expression of HMG-CoA reductase and increasing LDL receptor and Sp1 expression in a mammal.

[0026] Our surprising finding that lunasin inhibits H3 acetylation, reduces expression of HMG-CoA reductase and increases LDL receptor and Sp1 expression in a mammal can be used for numerous health related benefits, including but not limited to, to lower total or LDL cholesterol levels or to prevent, control or treat cancers in mammals.

These effects of lunasin can be further increased by developing formulations of lunasin and lunasin derivatives that are optimized for adsorption and delivery to the liver. [0027] Lunasin is the small subunit peptide of a cotyledon-specific 2S albumin. Fig. 1 shows the 2S albumin protein and the small lunasin subunit. It has been shown that constitutive expression of the lunasin gene in mammalian cells disturbs kinetochore formation and disrupts mitosis, leading to cell death (2). When applied exogenously in mammalian cell culture, the lunasin peptide suppresses transformation of normal cells to cancerous foci that are induced by chemical carcinogens and oncogenes. To elucidate its chemopreventive mechanism of action, we have shown that lunasin (a) is internalized through its RGD cell adhesion motif, (b) colocalizes with hypoacetylated chromatin in telomeres at prometaphase, (c) binds preferentially to deacetylated histone H4, which is facilitated by the presence of a structurally conserved helical motif found in other chromatin-binding proteins, (d) inhibits histone H3 and H4 acetylation, and (e) induces apoptosis in E1 A-transfected cells (4). Based on these results, a novel chemopreventive mechanism has been proposed wherein lunasin gets inside the nucleus, binds to deacetylated histones, prevents their acetylation and inhibits gene expression like those controlled by the Rb tumor suppressor and h-ras oncogene.

[0028] Lunasin's Effect On Expression Of Sp1 Coactivator

[0029] Inhibition of H3 histone acetylation by PCAF histone acetylase enzyme is required for the SREBP activation of genes involved in cholesterol biosynthesis including HMG-CoA reductase (9). Previous study has shown that lunasin is a potent inhibitor of histone H3 acetylation in mammalian cells exposed to the histone deacetylase inhibitor, sodium butyrate (NaB) (4). To determine the effect of lunasin on histone H3 acetylation by PCAF, HAT assay reaction using acid-extracted histones from untreated HeLa cells as template was conducted, lmmunoblotted reaction products have been stained with antibodies against diacetylated histone H3 (Ac-Lys9 + Ac-Lys14) and the details of our experiment and its results are shown and described in Fig. 5. In brief, the HAT enzyme, PCAF, is shown to increase significantly histone H3 acetylation in the absence of lunasin (35-fold increase). However, the addition of lunasin in the PCAF reaction, resulted in dramatic reduction of histone H3 acetylation, indicating that lunasin is a potent inhibitor of histone H3 acetylation catalyzed by the PCAF acetylase enzyme.

[0030] To determine the specific lysine residue in histone H3 that is inhibited by lunasin from being acetylated, immunoblotted products of PCAF acetylase reactions were hybridized with antibodies raised against acetylated Lys 9 and acetylated Lys 14 in H3 terminal tails. The results and details of our experiments, as shown and described in Fig. 6, demonstrate that lunasin specifically binds to Lys 14, preventing it from being acetylated by PCAF.

[0031] In one exemplary embodiment of the present invention, a method of inhibiting H3 acetylation in a mammal is provided. The method includes providing an effective amount of lunasin peptides to a mammal to inhibit H3 acetylation in the mammal.

[0032] In another exemplary embodiment of the present invention, a method of reducing expression of HMG-CoA reductase in a mammal is provided. The method includes providing an effective amount of lunasin peptides to a mammal to reduce expression of HMG-CoA reductase in the mammal.

[0033] In yet another exemplary embodiment of the present invention, a method of increasing LDL receptor expression in a mammal is provided. The method includes providing an effective amount of lunasin peptides to a mammal to increase LDL receptor expression in the mammal. [0034] In yet another exemplary embodiment of the present invention, a method of increasing Sp1 transcriptional activator expression in a mammal is provided. The method includes providing an effective amount of lunasin peptides to a mammal to increase Sp1 transcriptional activator expression in the mammal.

[0035] In one aspect of at least one embodiment of the present invention, the effective amount of lunasin peptides that inhibit H3 acetylation, reduce expression of HMG-CoA reductase, increase LDL receptor expression or increases Sp1 transcriptional activator expression in a mammal is 25 to 100 mgs daily. It should be appreciated that the effective amount of lunasin will depend, at least in part, on the size, weight, health and desired goals of the mammals consuming the compositions. Accordingly, it is believed that in at least one embodiment, the effective amount of lunasin provided to the mammal is 25 mg to 100 mg daily.

[0036] In another aspect of at least one embodiment of the present invention, the lunasin peptides include lunasin peptides or lunasin peptide derivatives. It should also be appreciated that the present invention includes the use of lunasin peptide derivatives, which are any peptides that contain the same functional units as lunasin. It should also be appreciated the products and compositions of the present invention can be used in, foods, powders, bars, capsules, shakes and other well known products consumed by mammals or used separately.

[0037] In yet another aspect of at least one embodiment of the present invention, the lunasin peptides are obtained from, soy, seed bearing plants other than soy, using recombinant DNA techniques and synthetic polypeptide production or any combination thereof.

[0038] In yet another aspect of at least one embodiment of the present invention, the method includes providing an effective amount of one or more protease enzyme inhibitors with or without the lunasin peptides. The protease enzyme inhibitors act to protect lunasin from digestion and facilitate absorption and delivery to the appropriate target areas. Examples of appropriate protease enzyme inhibitors include, but are not limited to, pancreatin, trypsin and/or chymotrypsin inhibitors. It should be appreciated that the scope of the present inventions includes the use of the lunasin and/or lunasin derivatives with any other composition or product that is known or believed to facilitate lunasin's absorption or delivery in a mammal.

[0039] While the products, compositions and related methods have been described in terms of what are presently considered to be the most practical and preferred embodiments, it is to be understood that the disclosure need not be limited to the disclosed embodiments. It is intended to cover various modifications and similar arrangements included within the spirit and scope of the claims, the scope of which should be accorded the broadest interpretation so as to encompass all such modifications and similar structures. The present disclosure includes any and all embodiments of the following claims. All the patents, journal articles and other documents discussed or cited above are herein incorporated by reference. [004O] EXAMPLES

[0041] The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

EXAMPLE 1 [0042] Lunasin Reduces Expression of HMG-CoA Reductase, Increases Expression of LDL Receptor. [0043] The lowering of serum cholesterol by statin drugs is achieved by competitively inhibiting the HMG-CoA reductase, the rate limiting enzyme in the body's metabolic pathway for synthesis of cholesterol. By reducing endogenous cholesterol synthesis, statins also cause liver cells to up regulate expression of the LDL receptor, leading to increased clearance of low-density lipoprotein (LDL) from the bloodstream (9). In 1985, Michael Brown and Joseph Goldstein received the Nobel Prize in Medicine for their work in clarifying this LDL-lowering mechanism.

[0044] Transcriptional regulation of HMG-CoA reductase and LDL receptor is controlled by the Sterol Regulatory Element-Binding Protein -1 and -2 (SREBP). This protein binds to the sterol regulatory element (SRE) located on the 5' end of the reductase and the LDL receptor genes. When SREBP is inactive, it is bound to the ER or nuclear membrane. When cholesterol levels fall, SREBP is released from the membrane by proteolysis and migrates to the nucleus, where it binds to the SRE to up regulate transcription of HMG-CoA reductase and LDL receptor (8, 9). [0045] In cell culture of HepG2 liver cells, it is possible to activate SREBP and increase the expression of HMG-CoA reductase and LDL-receptor by removing cholesterol in the growth media. This can be achieved by exposing the cells to serum-free media for 24 hours (15, 16).

[0046] The following related experiments were performed to evaluate the effect of lunasin on HMG-CoA reductase expression and LDL-receptor expression.

[0047] In the first experiment, HepG2 cells (1 x 10⁶) were treated with or without 10 uM synthetic lunasin in DMEM with 10% FBS for 24 hours before growth media was replaced with cholesterol-free media to activate SREBP. After 24 hours, total protein was extracted and 10 ug protein was loaded onto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane, and immunostained with primary antibodies raised against HMG-CoA reductase and actin (to show equal loading of proteins). Spot densitometer values are obtained by digital scanning and Un-Scan It software, and represent mean and standard deviation of data from three separate experiments. The results are shown in Figure 2. [0048] In the second experiment, HepG2 cells (1 x 10⁶) were treated with or without 10 uM synthetic lunasin in DMEM with 10% FBS for 24 hours before growth media is replaced with cholesterol-free media to activate SREBP. After 24 hours, total protein was extracted and 10 ug proteins loaded onto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane, and immunostained with primary antibodies raised against LDL-receptor and actin (to show equal loading of proteins). Spot densitometer values were obtained by digital scanning and Un-Scan It software, and represent mean and standard deviation of data from three separate experiments. The results are shown in Figure 3.

[0049] Figures 2 and 3 show the upregulation of HMG-CoA reductase (98% increase) and LDL-receptor (34% increase) when HepG2 cells are grown in cholesterol-free media for 24 hours. However when lunasin is added to the cholesterol-free media, the expression of the HMG-CoA reductase is reduced by more than 50% (Figure 2), while the expression of LDL-receptor has increased by more than 60% (Figure 3).

[0050] This effect of lunasin is similar to statin drugs that reduces endogenous cholesterol synthesis by inhibiting HMG-CoA reductase activity, which leads to increased LDL receptor expression. However, while it is not intended that the present invention be limited to any precise mechanism or mode of action, the mode of action of lunasin is believed to differ from statin drugs in that it appears to inhibit expression of HMG-CoA reductase at the transcriptional level, rather than on inhibiting its enzyme activity. Like statin drugs, lunasin up regulates the expression of LDL-receptor gene. Again, while it is not intended that the present invention be limited to any precise mechanism or mode of action, the contrasting effect of lunasin on these two SREBP-controlled genes can be explained by the selective recruitment of different co-regulatory transcription factors to two separate cholesterol-regulated promoter/regulatory sequences.

EXAMPLE 2 [0051] Lunasin's Effect On Expression Of Sp1 Coactivator

[0052] Unlike HMG-CoA reductase, SREBP activation of LDL-receptor by sterol depletion requires increased recruitment of Sp1 co-activator to a site adjacent to SREBP in the promoter/regulatory sequence of LDL-receptor gene (25). As shown in Figure 3, the up regulation of LDL-receptor by lunasin (LS) in cholesterol-free media may be due to increased availability and recruitment of the Sp1 coactivator to the LDL-receptor promoter/regulatory sequence. To test this hypothesis, the level of Sp1 was determined in lunasin-treated growth media and cholesterol-free media by Western analysis using Sp1 antibody, as follows: HepG2 cells (1 x 10⁶) were grown from confluence in DMEM with 10% FBS for 24 hours before growth media was replaced with fresh growth media or cholesterol-free media (to activate SREBP) and treated with, or without 10 uM synthetic lunasin. After 24 hours, total protein was extracted from each treatment and 10 ug protein loaded onto 10% Tris-glycine gels, electroblotted onto nitrocellulose membrane, and immunostained with primary antibodies raised against Sp1 and actin (to show equal loading of proteins). Spot densitometer values were obtained by digital scanning and Un-Scan It software and represent data from one experiment. The results are shown in Figure 4. [0053] Figure 4 shows that Sp1 levels in control and lunasin-treated growth media were not significantly different. However, Sp1 levels increased in cholesterol-free media by 23%, compared to the growth media. The addition of lunasin in the cholesterol-free media further increased Sp1 levels by almost 60%, which closely mirrors the increase in LDL-receptor levels in lunasin-treated, cholesterol-free media. [0054] The data from these experiments indicate that the increase in LDL-receptor expression by lunasin in sterol-depleted media could be attributed to the increased availability of the Sp1 transcriptional co-activator. Also, the inhibition of HMG-CoA reductase expression by lunasin lowers intracellular cholesterol levels that keep SREBP activated, resulting in the upregulation of LDL receptor expression. [0055] Therefore, the data shows that lunasin inhibits the expression of HMG-CoA reductase, the rate limiting enzyme in the body's metabolic pathway for synthesis of cholesterol, and at the same time increases the expression of the LDL receptor, leading to increased clearance of low-density lipoprotein (LDL) from the bloodstream, which will lower total and LDL cholesterol in a mammal. [0056] Most circulating cholesterol in mammals is synthesized internally, on average 1000 mg/day compared to 200-300 mg/day from intestinal intake in a human diet. Thus the internal production of cholesterol, as catalyzed by HMG-CoA reductase and the amount of LDL receptors in liver cell membranes, is the single most important factor in modulating cholesterol levels in mammals. Accordingly, these experiments demonstrate that an effective amount of lunasin reduces both LDL and total cholesterol levels in a mammal. EXAMPLE 3

[0057] Lunasin can be extracted from commercial sources of soy protein. Lunasin has been found in significant amounts from commercial sources of soy protein and its homologues from other seed sources such as barley and wheat. To identify preferred sources for the starting raw material that can be used for lunasin extraction, several commercially available soy protein products were screened for the presence of lunasin.

[0058] The procedure used was as follows: approximately 500 mg of soy protein samples obtained from different commercial sources (Solae, St. Louis, MO) were dissolved in 50 ml_ of aqueous phosphate buffer (pH 7.2) by shaking for 1 hour at room temperature. Samples were centrifuged at 2500 rpm for 30 minutes and the aqueous fraction separated and put in separate tubes. Protein concentrations were measured by Bradford assay and around 20 ug of total protein were loaded onto two Bio-Rad Laboratories (Hercules, California) 16% Tris-TNcine gels. One of the SDS-PAGE gels (I) was stained with Coomasie blue and destained before digital imaging. The 5 kDa lunasin band is indicated by arrow. The other (II) is electroblotted onto nitrocellulose membrane and incubated with affinity-purified lunasin polyclonal antibody (Pacific Immunology, Ramona, California) followed by HRP-conjugated donkey anti-rabbit secondary antibody (Amersham Biosciences, Piscataway, New Jersey). Lunasin immunosignals (indicated by arrow) are detected using the ECL Western blotting kit from Amersham.

[0059] The results showed that lunasin concentration varies dramatically from source to source. This assay is a useful tool in identifying sources of natural lunasin for use in the compositions and methods of the present invention. The soy concentrate that contained the most lunasin was used as a starting material in a buffer extraction procedure to produce the lunasin-enriched soy concentrate (LeSC) used in the following experiments.

EXAMPLE 4

[0060] Formulated lunasin-enriched soy concentrate (LeSC) and LeSC supplemented with soy flour (SF) contain significant amounts of lunasin. This experiment evaluated the amount of lunasin in lunasin-enriched soy concentrate (LeSC) and LeSC supplemented with soy flour. [0061] Lunasin-enriched soy concentrate was produced by first identifying commercially available soy protein preparations that contain significant amounts of lunasin by Western blot analysis using lunasin polyclonal antibody, as described in Example 3. The soy protein concentrate identified to contain the most lunasin was used as starting material in a one-step buffer extraction procedure (0.1 X PBS pH 7.2) followed by centrifugation to separate the supernatant. Two volumes of acetone were added to supernatant and precipitate was separated by centrifugation with filter bags before vacuum drying to get the lunasin-enriched soy concentrate.

[0062] Efforts to make lunasin more resistant to undesired excessive digestion, improve its bioavailability, and retain its bioactivity when ingested, resulted in the discovery of at least one of the preferred embodiments of the present invention, a composition comprising lunasin enriched soy concentrate and soy flour.

[0063] In at least one embodiment of the present invention, compositions of the present invention that comprise naturally derived lunasin can be optimized for use in particular methods of the present invention by varying the amount of total protein and lunasin content, which can be controlled by the amount of soy concentrate used, and varying the amount of lunasin protection from digestion, which can be controlled by the amount of minimally heated soy flour used.

[0064] For food based items it is sometimes desirable to limit the amount of protease inhibitors in a product. For example, U.S. Patent Application No. 20070092633, filed April 26, 2007, hereby incorporated by reference, teaches that part of the standard processing of some soy products includes heat treatment to inactivate anti-nutritional elements such as Bowman-Birk and Kuntz inhibitors. Therefore, in a preferred embodiment of the present invention, a composition comprising lunasin and soy flour is optimized through preparation methods describe herein or known to one skilled in the art, to have a level of protease inhibitors sufficient to protect lunasin biological activity during digestion but not sufficient to have levels of anti-nutritional elements that are undesirable for oral use.

[0065] Clinical trials on a 50:50 blend of soy concentrate and soy flour led to a 20-30% reduction of LDL cholesterol (17, 18.) Those clinical trials were performed without the knowledge that lunasin is an active element in soy concentrate in reducing LDL cholesterol, and therefore did not control for the level of lunasin present in the blend. The present invention teaches improved methods of determining lunasin concentration in starting materials and final products of the present invention, so as to maximize the concentration of lunasin and therefore the activity of compositions of treatment in cholesterol related applications. In at least one preferred embodiment of the present invention the ratio of soy flour to soy concentrate is between 10:90 and 50:50, more preferably between 20:80 and 40:60, more preferably approximately 30:70 soy floursoy concentrate. This ratio for minimally heated soy flour and soy concentrate was determined to provide a biologically active concentration of lunasin and as well as sufficient protection from digestion by the soy flour.

[0066] In the following several experiments, minimally heated soy flour (SF) was added to the starting soy concentrate (at a 30:70 w/w mixture) before buffer extraction with 0.1 x PBS pH 7.2 and acetone precipitation to produce lunasin enriched soy concentrate plus soy flour (LeSC + SF.)

[0067] The Western blotting analysis procedure used in this experiment was as follows: approximately 20 ug of total protein from LeSC, SF and the LeSC+SF were electrophoresed in 16% Tris-TNcine gels and electroblotted onto nitrocellulose membrane. Blots were incubated with lunasin polylconal antibody followed by HRP- conjugated anti-rabbit secondary antibody before lunasin immunosignals were detected with the ECL kit. The results showed that both LeSC and LeSC + SF contained significant amounts of lunasin.

EXAMPLE 5

[0068] Lunasin-enriched soy concentrate with soy flour (LeSC + SF) retains bioactivity even when digested with digestive enzymes. Biological activity of LeSC (A), LeSC + SF (B), digested LeSC + SF (C), digested LeSC (D), digested soy protein isolate (E) and digested soy concentrate (F) was measured using the H3 histone acetyltransferase (HAT) assay (see Example 8.) Around 100 mg total protein of LeSC, LeSC + SF, soy protein isolate and soy concentrate were digested by mixing pancreatin (Sigma Life Sciences, Saint Louis, Missouri) at 1 :1 (w/w) and incubating for 30 min. at 4O⁰C. To confirm that the HAT assay is working, treatment with synthetic lunasin (+synl_) was included. Synthetic lunasin reduced acetylation of histone H3 by the histone acetylase enzyme, PCAF, using core histones isolated from chicken erythrocyte (Upstate/Millipore, Billerica, MA) as template for the HAT assay. Around 10 ug of sample protein was incubated with 1 ug of core histones before undergoing HAT reaction with PCAF enzyme and acetyl CoA substrate. Reaction products were run on 16% Tris-Tricine gels and electroblotted onto nitrocellulose membrane. Blots were incubated with primary antibody raised against acetylated H3 (diacetylated at histone14 and histonel O) and HRP-conjugated anti-rabbit secondary antibody before detecting signals using the ECL kit. Low signals indicated that the lunasin peptide was bioactive because it prevented the acetylation of histone H3. Strong signals indicated that the lunasin peptide had been digested and rendered inactive, thus failing to impact levels of histone H3 acetylation.

[0069] There was significant reduction in H3 acetylation in the presence of synthetic lunasin compared to the untreated control. Both the LeSC and the LeSC + SF were able to significantly reduce H3 acetylation by PCAF, indicating that the lunasin found in both soy protein extracts is biologically active. Pancreatin digestion of LeSC + SF reduced the biological activity but not to the extent observed when LeSC alone is digested. Like LeSC, soy protein isolate and soy concentrate that contain significant amounts of lunasin, did not show lunasin biological activity after pancreatin digestion

These results indicate that the formulated LeSC + SF protects lunasin to a certain degree from pancreatin digestion, and allows lunasin to retain its biological activity.

EXAMPLE 6

[0070] Partial digestion of formulated LeSC + SF increases biological activity of lunasin. A confirmatory experiment to determine the biological activity of digested and undigested LeSC and LeSC + SF was conducted using a different core histone template. This time we used the core histones extracted from HeLa tumor cells. Unlike the chicken erythrocyte cells, core histones from sodium butyrate treated HeLa cells are commercially available (Upstate/Millipore, Piscataway, NJ), and can be used as a positive control for histone acetylation. The core histones isolated from untreated HeLa cells were used as a negative control (low levels of histone acetylation) and as template for the HAT assay.

[0071] The HAT bioactivity assay was conducted using acid extracted core histones from HeLa cells (Upstate/Millipore) as a template (temp (-) control) for the PCAF catalyzed HAT reaction. Core histones from sodium butyrate (NaB) treated HeLa cells were used as a positive control since NaB is a histone deacetylase inhibitor known to increase histone acetylation. The inhibitory effect of synthetic lunasin (+synL) on histone H3 acetylation by PCAF was used to compare the effect of lunasin-enriched soy concentrate (A), digested LeSC (A dig), LeSC + SF (B) and digested LeSC + SF (B dig). LeSC and LeSC + SF were partially digested by adding pancreatin at 1 :0.5 (w/w) and incubating at 38⁰C for 15 min. The numbers below the legend indicate relative densitometer readings normalized using immunosignal from the template (temp). Low numbers indicate presence of lunasin biological activity.

[0072] The results showed that significant reduction in H3 acetylation in the presence of synthetic lunasin was seen. The undigested LeSC (A) and LeSC + SF (B) showed reduced levels of H3 acetylation, indicating that the natural lunasin found in these soy extracts was biologically active. Partial digestion of LeSC (A Dig) led to the loss of biological activity.

[0073] Surprisingly, partial digestion of LeSC +SF resulted in an increase in biological activity rather than a decrease. While it is not intended that the present invention be limited to any precise mechanism, it is believed that lunasin is covalently bound to high molecular weight protein complexes and that, with the protection of soy flour, partial digestion only breaks down these bonds and releases, but does not destroy, bioactive lunasin into the solution. In a preferred embodiment of the present invention, lunasin is partially digested prior to use. In another preferred embodiment of the present invention, soy flour is present when lunasin is partially digested. [0074] LeSC + SF was partially digested by mixing it with freshly prepared pancreatin solution (10 ug/mL of distilled water) in a 1 :0.5, (w/w) ratio. Mixture was incubated at 38⁰C for 15 min. before proteases and digestive enzymes were inactivated by boiling for 5 min and then quenching in ice. Under these digestion conditions the lunasin in the LeSC soy extract was digested and inactivated while that of LeSC + SF were more biologically active. However, the conditions for the partial digestion of LeSC + SF has to be determined empirically by analyzing digestion products for lunasin content and biological activity using the HAT assay.

[0075] Variations in the sources of pancreatin and protease enzymes, the age of the protease enzyme, or incubation conditions can lead to variability in digestion conditions. For example, the use of one month old preparations of pancreatin for partial digestion led to the degradation and loss of activity of lunasin under similar incubation conditions described above. Therefore, in a preferred embodiment of the present invention, acceptable ranges for concentration of and incubation time with the protease enzymes are determined using an assay such as the HAT assay used above to evaluate biological activity of the treated compositions.

EXAMPLE 7

[0076] Chymotrypsin inhibitors (Chy) protect the bioactivity of lunasin. To determine which protease inhibitors found in soy protects lunasin from digestion, soybean trypsin inhibitor and trypsin + chymotrypsin inhibitors were obtained from Sigma and mixed with LeSC on 1 :1 w/w ratio. The mixtures were digested with pancreatin, and digestion products immunostained with lunasin antibody.

[0077] Details of the experiment are as follows. LeSC + soybean trypsin inhibitors (1 :1 w/w) (Sigma) and LeSC + trypsin and chymotrypsin inhibitors (1 :1 w/w) (Sigma) were digested with pancreatin (1 :1 w/w) by incubating at 38⁰C for 15 min. Digestion products and LeSC were analyzed by Western blot analysis using lunasin primary antibody and synthetic lunasin as standard controls.

[0078] HAT bioactivity assay was conducted using core histones from chicken erythrocyte cells (Upstate/Millipore) as a template for the PCAF catalyzed HAT reaction. The inhibitory effect of synthetic lunasin (+synL) on histone H3 acetylation by PCAF as compared to the negative untreated control (- synL) was used to compare the effect of digested LeSC (A), digested LeSC + try + chy (B), digested LeSC + try (C), undigested LeSC (D) and undigested LeSC +SF (E.) [0079] The results showed that in the LeSC ÷trypsin + chymotrypsin inhibitors sample lunasin was better protected from digestion than in the LeSC ÷trypsin inhibitor sample. Likewise, in HAT assays to determine lunasin biological activity, digestion of LeSC + trypsin + chymotrypsin inhibitors was significantly more bioactive than LeSC + trypsin inhibitor. Pancreatin digestion of LeSC led to the loss of biological activity. These results indicate that the presence of chymotrypsin inhibitors in lunasin-enriched soy concentrate (LeSC) both helps protect the biological activity of lunasin and helps protect lunasin from excessive digestion.

EXAMPLE 8

[0080] Screening Assay to determine lunasin biological activity. [0081] Core histones purified from chicken erythrocyte cells were used as templates in histone acetylase (HAT) reactions using PCAF histone acetylase enzyme, in the presence or absence of around 2-10 uM lunasin. The core histone template and lunasin-enriched soy concentrates (LeSC and LeSC + SF) were mixed (10:1 w/w) and incubated in ice for 5 min and 25⁰C for 10 min before mixture was added to 1 X HAT reaction mix, 1 uM acetyl CoA and 5 uL PCAF (based on recommended concentration from Upstate/Millipore). Reaction mixture was incubated at 3O⁰C while shaking at 250 rpm for 1 h. Reaction was stopped by adding Laemmli stop buffer (1 :1 v/v) with beta- mercaptoethanol, and boiling for 5 min. before quenching in ice for 15 min. The products of PCAF HAT reaction were run on 16% SDS-PAGE, blotted onto nitrocellulose membrane and immunostained with primary antibodies raised against diacetylated histone H3 (Ac-Lys 13 + Ac-Lys14 H3) followed by HRP-conjugated anti- rabbit secondary antibody. Chemiluminescent signals from antibody complexes were visualized using standard chemiluminescent reagents and exposed to Kodak BioMAX film, developed and spot densitometer measured by using digital scanner and UN- SCAN-IT software program from Silk Scientific (Orem, Utah). [0082] The results showed the reduction of H3 acetylation in the reaction mixtures treated with LeSc and LeSC + SF as compared to the untreated control, indicating that this screening procedure can determine the biological activity of lunasin-enriched soy concentrates. It was also determined that digestion of LeSC eliminates biological activity but not that of LeSC + SF which shows only a partial reduction of biological activity.

EXAMPLE 9

[0083] The in vivo activity of the presently described compositions, as well as treatment utilization of kits and treatment methods, may be optionally determined by either of the following procedures. [0084] Male dogs (beagles, ranging from about 9 to about 14 kilograms, 1 to 4 years old) are fed a standard dog feed supplemented with 5.5% lard and 1 % cholesterol. Baseline blood samples are drawn from fasted dogs prior to initiating the study to obtain reference values for plasma cholesterol. Dogs are then randomized to groups of five animals with similar plasma cholesterol levels. The animals are dosed in accordance with a treatment method described herein immediately prior to diet presentation for seven days. Blood samples are obtained 24 hours after the last dose for plasma cholesterol determinations. Plasma cholesterol levels are determined by a modification of the cholesterol oxidase method using a commercially available kit.

[0085] In an optional alternative procedure, hamsters are separated into groups of six and given a controlled cholesterol diet containing 0.5% cholesterol for seven days. Diet consumption is monitored to determine dietary cholesterol exposure. The animals are dosed in accordance with a treatment method described herein once daily beginning with the initiation of diet. Dosing is by oral gavage. All animals moribund or in poor physical condition are euthanized. After seven days, the animals are anesthetized by intramuscular (IM) injection of ketamine and sacrificed by decapitation. Blood is collected into vacutainer tubes containing EDTA for plasma lipid analysis and the liver is excised for tissue lipid analysis. Lipid analysis is conducted as per published procedures (e.g., Schnitzer-Polokoff et al., Comp. Biochem. Physiol., 99A, 4 (1991 ), pp. 665-670 and data is recorded as percent reduction of lipid versus control.

[0086] References [0087] The numeric references incorporated above correspond to the following list of published papers and abstracts. All of the below listed publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

1 . Galvez, A.F., Revilleza, M. J. R. & de Lumen, B. O. A novel methionine-rich protein from soybean cotyledon: cloning and characterization of cDNA. Plant Physiol 114:1567 (1997).

2. Galvez, A.F. & de Lumen, B. O. A soybean cDNA encoding a chromatin-binding peptide inhibits mitosis of mammalian cells. Nature Biotech. 17: 495-500 (1999).

3. de Mejia EG, Vasconez M., de Lumen BO & Nelson R. Lunasin concentration in different soybean genotypes, commercial soy protein and isoflavone products. J Agric Food Chem 52: 5882-5887 (2004).

4. Galvez, A.F. Chen, N., Macasieb, J., & de Lumen, B. O. Chemopreventive property of a soybean peptide. Cancer Res. 617473-7478 (2001 ).

5. De Pinho, R.A. The cancer-chromatin connection. Nature 391 : 533-536 (1998). 6. Kuzmin I & GeN L. DNA methylation and chromatin modifications in cancer and development, lnt Arch Biosci 2001 : 1047-1056 (2001 ).

7. Magbanua M, Dawson K, Huang L, Malyj W, Gregg J, Galvez A & Rodriguez RL. Nutrient - Gene Interactions Involving Soy Peptide and Chemopreventive Genes in Prostate Epithelial Cells, in Nutritional Genomics - Discovering the Path to

Personalized Nutrition, J. Kaput and R. L. Rodriguez eds., Wiley and Sons, New Jersey (2005).

8. Bennett MK & Osborne TF. Nutrient regulation of gene expression by the sterol regulatory element binding proteins: Increased recruitment of gene-specific coregulatory factors and selective hyperacetylation of histone H3 in vivo. PNAS 97:

6340-6344 (2000).

9. Brown MS & Goldstein JL. Lowering plasma cholesterol by raising LDL receptors. Atherosclerosis Suppl δ: 57-59 (2004).

10. Sirtori CR, Gatti E, Mantero O, Conti F., et al. Clinical experience with the soybean protein diet in the treatment of hypercholesterolemia. Am J Clin Nutr. 32:1645-1658

(1979).

1 1 . Descovich GC, Ceredi C, Gaddi A., Benassi MS, et al., Multicentre study of soybean protein diet for outpatient hyper-cholesterolaemic patients. Lancet 2:709- 712 (1980). 12. Lam, Y., Galvez, A., and de Lumen, B. O. Lunasin suppresses E1 A-mediated transformation of mammalian cells but does not inhibit growth of immortalized and established cancer cell lines. Nutrition & Cancer, 47/ 88-94 (2003). 13. Coqueret, O. New roles for p21 and p27 cell-cycle inhibitors: A function for each cell compartment? Trends in Cell Biology, 13: 65-70, (2003). 14. Bruzzone, R., White, T. W., and Paul, D. L. Connections with connexins: The molecular basis of direct intercellular signaling. European Journal of Biochemistry, 238: 1 -27 (1996).

15. Mullen E, Brown RM, Osborne TF & Shay NF. Soy isoflavones affect sterol regulatory element binding proteins (SREBPs) and SREBP-regulated genes in HepG2 cells. J. Nutr. 134: 2942-2947 (2004).

16. Gherardi E., Thomas K, Le Cras TD, Fitzsimmons C, Moorby CD & Bowyer DE. Growth requirements and expression of LDL receptor and HMG-CoA reductase in HepG2 hepatoblastoma cells cultured in a chemically defined medium. J Cell Sci. 103:531 -539 (1992).

17.Sirtori CR, Gatti E, Mantero O, Conti F., et al. Clinical experience with the soybean protein diet in the treatment of hypercholesterolemia. Am J Clin Nutr. 32:1645-1658 (1979).

18.Descovich GC, Ceredi C, Gaddi A., Benassi MS, et al., Multicentre study of soybean protein diet for outpatient hyper-cholesterolaemic patients. Lancet 2:709-712 (1980).

Claims

I CLAIM:

1 . A method of inhibiting H3 acetylation by PCAF in a mammal, the method comprising: providing an effective amount of lunasin peptides to a mammal to inhibit PCAF from acetylating histone H3 in the mammal.

2. A method of claim 1 , wherein the effective amount of lunasin peptides that inhibits PCAF from acetylating H3 in a mammal is between 25 and 100 mgs daily.

3. A method of claim 1 , wherein the lunasin peptides include lunasin peptides or lunasin peptide derivatives.

4. A method of claim 1 , wherein the lunasin peptides are obtained from one or more of the following groups: soy, seed bearing plants other than soy, using recombinant DNA techniques and synthetic polypeptide production.

5. A method of claim 1 , further comprising:

providing an effective amount of one or more protease enzyme inhibitors.

6. A method of reducing expression of HMG-CoA reductase in a mammal, the method comprising: providing an effective amount of lunasin peptides to a mammal to reduce expression of HMG-CoA reductase in the mammal.

7. A method of claim 6, wherein the effective amount of lunasin peptides that reduces expression of HMG-CoA reductase in a mammal is 25 to 100 mgs daily.

8. A method of claim 6, wherein the lunasin peptides include lunasin peptides or lunasin peptide derivatives.

9. A method of claim 6, wherein the lunasin peptides are obtained from one or more of the following groups: soy, seed bearing plants other than soy, using recombinant DNA techniques and synthetic polypeptide production.

10. A method of claim 6, further comprising:

providing an effective amount of one or more protease enzyme inhibitors.

1 1 . A method of increasing LDL receptor expression in a mammal, the method comprising: providing an effective amount of lunasin peptides to a mammal to increase LDL receptor expression in the mammal.

12. A method of claim 1 1 , wherein the effective amount of lunasin peptides that increases LDL receptor expression in a mammal is 25 to 100 mgs daily.

13. A method of claim 1 1 , wherein the lunasin peptides include lunasin peptides or lunasin peptide derivatives.

14. A method of claim 1 1 , wherein the lunasin peptides are obtained from one or more of the following groups: soy, seed bearing plants other than soy, using recombinant DNA techniques and synthetic polypeptide production.

15. A method of claim 1 1 , further comprising:

providing an effective amount of one or more protease enzyme inhibitors.

16. A method of increasing Sp1 transcriptional activator expression in a mammal, the method comprising: providing an effective amount of lunasin peptides to a mammal to increase Sp1 transcriptional activator expression in the mammal.

17. A method of claim 16, wherein the effective amount of lunasin peptides that increases Sp1 transcriptional activator expression in a mammal is 25 to 100 mgs daily.

18. A method of claim 16, wherein the lunasin peptides include lunasin peptides or lunasin peptide derivatives.

19. A method of claim 16, wherein the lunasin peptides are obtained from one or more of the following groups: soy, seed bearing plants other than soy, using recombinant DNA techniques and synthetic polypeptide production.

20. A method of claim 16, further comprising:

providing an effective amount of one or more protease enzyme inhibitors.