1 Updating the research on the chemopreventive and therapeutic role of
2 peptide lunasin
3 Running title: Chemopreventive and therapeutic properties of peptide lunasin
4 Chia-Chien Hsieh1, Cristina Martínez-Villaluenga2, Ben O. de Lumen3, Blanca Hernández-
5 Ledesma4*
6
7 1 Department of Human Development and Family Studies (Nutritional Science & Education),
8 National Taiwan Normal University, No. 162, Heping East Road, Section 1, Taipei 10610,
9 Taiwan
10 2 Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Juan de la Cierva 3,
11 28006 Madrid
12 3 Department of Nutritional Science and Toxicology, University of California at Berkeley,
13 119 Morgan Hall, Berkeley, CA 94720-3104, USA
14 4 Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM, CEI
15 UAM+CSIC). Nicolás Cabrera, 9. 28049 Madrid, Spain
16 17 * Corresponding author: B. Hernández-Ledesma
18 Nicolás Cabrera, 9. 28049 Madrid, Spain
19 Phone: +34 910017970
20 Fax: +34 910017905
21 e-mail: [email protected]
22 1
23 Abstract
24 Chronic diseases have become the medical challenge of the 21st century because of their high
25 incidence and mortality rates. Modulation of diet and lifestyle habits is considered as the best
26 strategy for the prevention of these disorders. Health promoting benefits beyond their
27 nutritional effects have been described for multiple dietary compounds. Among these
28 compounds, peptide lunasin is considered as one of the most promising. Naturally present in
29 soybean, lunasin has been extensively studied in the last two decades because of its potential
30 against chronic diseases, such as cancer, cardiovascular and immunological disorders. The
31 purpose of this article is to summarize the evidence on the presence of lunasin in soybean and
32 derived foods, and its bioavailability once it is orally ingested. The protective and therapeutic
33 effects of this peptide against cancer, oxidative stress, inflammation, and high cholesterol
34 levels as well as the molecular mechanisms of action involved in these effects are also
35 described in this review.
36
37 Keywords: lunasin; bioactive peptide; chemopreventive and therapeutic properties; cancer;
38 chronic diseases
39
2
40 INTRODUCTION
41 Twenty-year history of peptide lunasin
42 Lunasin (from the Tagalog word “lunas” for cure) is a peptide identified in the
43 soybean cotyledon twenty years ago.1 It corresponds to a small subunit peptide encoded
44 within the soybean 2S albumin Gm2S-1 gene that also codes for a methionine-rich protein, a
45 signal peptide, and a linker peptide.2 Early studies carried out with the chimeric gene
46 encoding lunasin and tagged with the green fluorescent protein demonstrated that its
47 transiently transfection into murine embryo fibroblast C3H 10T1/2, hepatoma Hepa1c1c7
48 cells, and human breast cancer MCF-7 cells resulted in cell division arrest causing abnormal
49 spindle fiber elongation, chromosomal fragmentation, and cell lysis.3 These results suggested
50 that the binding of lunasin to chromatin prevented the normal formation of the kinetochore
51 complex in the centromere, thus leading to the disruption of mitosis, and eventually to cell
52 death. According to the first reports, lunasin identified in soybean contains 43 amino acids. Its
53 sequence (SKWQHQQDSCRKQLQGVNLTPCEKHIMEKIQGRGDDDDDDDDD, National
54 Center for Biotechnology Information, NCBI, accession number AAP62458) presents nine
55 aspartic acid (D) residues at the C-terminal end responsible for lunasin’s colocalization with
56 hypoacetylated chromatin through ionic interactions and for inhibition of histone H3
57 acetylation, the 33RGD35 cell adhesion motif responsible for lunasin adhesion and
58 internalization into cells, a predicted helical region (23E-I30) with homology to a conserved
59 region in chromatin-binding proteins, and twenty two residues at N-terminal facilitating the
60 binding to deacetylated N-terminal tail of histone H4.4 A molecular dynamics study was
61 carried out to characterize the exact three-dimensional structure of lunasin.5 It was revealed
62 that this peptide exhibits three separate α-helical bundles in its structure supported by residues
63 5H-C10, 22C-I30 and 35D-D41. Moreover, the last residues present in both N- and C-terminus
64 remain extended or unstructured, and the motif RGD plays a key role of hinge winding and 3
65 unwinding the central and C-terminus helical regions of the peptide. This study suggested that
66 the α-helix associated with aspartic acid (D) residues at C-terminal of lunasin plays a
67 recognition role in its binding with the chromatin residue and, could thus be responsible for its
68 anti-mitotic action in mammalian cell lines. Later on, a structural analysis by circular
69 dichroism at 25°C has concluded that lunasin contains 29% α-helix, 28% β-strands, 23%
70 turns, and 20% unordered.6 More recently, nuclear magnetic resonance and circular dichroism
71 have revealed that lunasin can adopt a reduced form with thiols or an oxidized form with
72 intramolecular disulphide bonds, both containing two helical segments (3W-K12 and 21P-I30)
73 and a single region with and extended β-sheet conformation corresponding to C-terminal
74 poly-D tail.7 Additionally, these authors found that these secondary structures are in
75 equilibrium with unstructured conformations.
76 Although the first studies characterized lunasin as a 43 amino acid peptide, the
77 analysis carried out in the last five years show controversial results. Seber and coworkers
78 purified lunasin from soybean white flake by anion exchange chromatography followed by
79 reversed-phase chromatography.8 The purified ≈14 kDa protein complex containing lunasin
80 was analyzed by electrospray ionization (ESI) mass spectrometry (MS) revealing that the
81 major peptide in this preparation had a monoisotopic mass of 5139.25, which is 114.02 Da
82 higher than the expected mass of 5025.23 Da for the 43 amino-acid form of lunasin described
83 in the literature. The mass difference suggested that the predominant form of purified lunasin
84 from soybean flake contained 44 amino acids with an additional asparagine (N) residue at C-
85 terminal. This fact was confirmed by the analysis of tryptic peptides derived from purified
86 lunasin by liquid chromatography (LC) coupled to tandem MS (LC-MS/MS). Also, a weak
87 signal with a monoisotopic mass 5025.23 Da was observed suggesting that a minor amount of
88 the 43 amino-acid form of lunasin may be present. Similar results have been recently obtained
89 by Serra et al. for lunasin purified from a commercial soybean beverage powder and 4
90 characterized by top-down proteomics.9 In addition to the unmodified 44-amino acids lunasin,
91 these authors found different post-translationally modified proteoforms resulting from diverse
92 degrees of glycation mainly at residue lysine (24K and 29K) as well the occurrence of
93 alternative side chain modifications, including oxidation (M), dihydroxy (K), dehydration
94 (D/N), deamidation (N/Q), methyl esterification (D), carbamylation (K), acetylation (K) and
95 pyro-glutamate conversion (N). All these modifications may have an important repercussion
96 on lunasin’s bioactive effects and its epigenetic regulatory capacity.9
97
98 Presence of lunasin in plants: what is its origin?
99 Since its discovery in soybean, studies have focused on purifying and quantifying
100 lunasin from different soybean varieties, reporting concentration values ranged from 1,100 to
101 14,000 g lunasin/g extracted protein or 500-810 g lunasin/g seed.10,11 The results obtained
102 from these studies demonstrate that the soybean genotype is the main affecting factor on
103 lunasin’s quantity in soybean seeds, indicating the possibility of selecting and breeding
104 varieties of soybean with higher content of this peptide.10 Environmental factors, mainly
105 temperature and soil moisture, have also been reported to affect lunasin’s concentration in
106 soybean seeds, whereas light and dark conditions do not seem to have any effect.12 Other
107 factors such as the stages of seed development and maturation have also a notable influence
108 on lunasin’s content in soybean. Seed maturation resulted in an important increase of this
109 content while sprouting leads to a continuing decrease of lunasin with soaking time.11
110 Germination time and temperature have significant influences on the composition and
111 concentration of nutrients and bioactive compounds such as lunasin in germinated soybean
112 flour from several Brazilian soybean cultivars.13-15
113 Once the biological activity of soybean lunasin was reported for the first time, a
114 number of studies focused on the search of this peptide in other plants. Its presence was 5
115 detected in both cereals and non-cereal plants with variable content. In cereals, this content
116 ranged from 13-99 μg g-1 seed in barley,16 200-300 μg g-1 seed in wheat,17 45-150 μg g-1 seed
117 in rye,18 429-6,458 μg g-1 grain in triticale,19 and 34-197 μg g-1 grain in oat.20 The content was
118 reported to be mainly influenced by the genotype.21 These authors found that the broad-sense
119 heritability for the lunasin content in this cereal was 70.4%. Lunasin production is also
120 dependent on the interaction of genotype and organic farming systems. Legzdina et al. found
121 that average amount of lunasin in barley was significantly higher in organically grown plants;
122 however, it is worth noting that some barley genotypes displayed the opposite effect.21 These
123 results provide the idea that it is possible to select genotypes that are appropriate for organic
124 farming and lunasin accumulation such as barley variety Rubiola. Lunasin has also been
125 detected in other plant sources, such as the black nightshade (Solanum nigrum L.)22 and the
126 pseudocereal amaranth (Amaranthus hypochondriacus L.)23 In amaranth, the lunasin peptide
127 is encrypted in a 22 kDa precursor protein, and the primary structure of the native precursor
128 protein differs from that of soybean lunasin.24 The sequence of amaranth lunasin-like peptide
129 (SKWQHQQDSCRKQLQGFKMTATPPCEKHITRAFRRAPIQQRGRGISTRRGDDDDDD-
130 DDD) showed high homology with the soybean peptide.
131 In the last years, studies have brought into question the origin of lunasin and its actual
132 presence in other plants than soybean. Mitchell and coworkers carried out an exhaustive
133 proteomic and transcriptomic analysis of wheat and other cereals, as well as a deeply search
134 in different DNA sequences databases that failed to identify sequences similar to those known
135 to encode peptide lunasin in soybean. 25 Similarly, Dinelly and co-workers, using chemical
136 (LC-MS) and molecular (polymerase chain reaction, PCR) analyses, reported the complete
137 absence of lunasin in the investigated wheat genotypes.26 These findings have been recently
138 supported by Alaswad and Krishman who conducted immunological analysis in cereals and
139 other plant species using polyclonal antibodies specific to the first 20 amino acid N-terminal 6
140 peptide and the 15 amino acid C-terminal peptide of lunasin.27 Results from this study
141 demonstrated that peptides identical to lunasin are absent in the studied plant species.
142 However, other proteins with higher molecular weight were recognized by soybean lunasin
143 antibodies providing the hypothesis that lunasin-like peptides could be released from
144 precursor proteins by the action of proteases from gastrointestinal or microbial origin. This
145 hypothesis is in line with the study of Rizzello and co-workers who investigated the presence
146 of lunasin-like polypeptides in nineteen traditional Italian legumes after sourdough
147 fermentation with Lactobacillus plantarum C48 and Lactobacillus brevis AM7, expressing
148 different peptidases.28 These authors confirmed the absence of lunasin in legume flours
149 although different lunasin-like polypeptides were found in bean and lentil varieties. After
150 lactic acid fermentation, legume sourdoughs showed increased number and intensities of
151 lunasin-like polypeptides, suggesting that endogenous legume proteases and microbial
152 peptidases were responsible for the release of lunasin-like fragments. Rizzello and co-workers
153 have additionally suggested a possible microbial origin for lunasin.29 According to these
154 authors, lunasin might be synthesized and secreted by microorganisms, either as a mature
155 peptide or a precursor which is further processed by a microbial or endogenous plant
156 proteinase. Further studies should be needed to elucidate the real origin of this peptide.
157
158 Lunasin in plant-derived products: extraction, identification, structure diversity and
159 production
160 Lunasin has been quantified in a variety of soybean derived products, foods and
161 beverages by using Western blot and ELISA assays.30,31 The concentration of this peptide in
162 those products is shown in Table 1. These methods require the use of antibodies that only
163 recognize the sequence corresponding to fragment 23EKHIMEKIQGRGDDDDD39, and thus,
164 they can suffer from cross-reactivity. Therefore, two dimensional analytical methods have 7
165 been applied combining an isoelectrofocusing separation with LC-MS and a time-of-flight
166 (TOF) analyzer for the selective and sensitive determination of lunasin in soybean-derived
167 products, such as textured soybean and soybean flour (Table 1).32 Recently, top-down
168 proteomic strategy was used to identify and characterize novel proteoforms of lunasin in
169 commercial soybean food products derived from food processing and natural chemical
170 reactions.9 Four different lunasin derived advanced glycation end products containing mainly
171 Nε-carboxy-methyl-lysine and Nε-carboxy-ethyl-lysine at 24K and 29K were detected.
172 In addition to the natural presence of lunasin in plant derived-foods, some processing
173 methodologies have been demonstrated to augment its concentration. Rizzello and others
174 revealed the potential role of microbial proteolytic system for increasing the lunasin
175 concentration in food matrices by sourdough fermentation.29 This study suggests new
176 alternatives of cost effective bio-production of lunasin over chemical synthesis as well as new
177 developments of functional fermented products containing this peptide.
178 In spite of the different beneficial properties that have been demonstrated for lunasin,
179 production of this peptide for commercial or research applications is very costly. In the last
180 years, efforts have been made to obtain lunasin in a higher concentrated or purified form.
181 Cavazos and others developed a method resulting in >90% purity of lunasin from defatted soy
182 flour.31 Simultaneously, Seber and others applied more advanced techniques, namely, anion-
183 exchange chromatography, ultrafiltration, and reversed-phase chromatography to purify
184 lunasin, resulting in >99% purity of lunasin from defatted soy flour.8 A recent work has been
185 published reporting the development of a novel method involving extraction of soybean flour
186 with 30% ethanol followed by preferential precipitation of lunasin and protease inhibitors by
187 calcium.33 This simple procedure yielded 3.2 g of lunasin-protease inhibitor concentrate from
188 100 g of soybean flour, and the entire isolation procedure was completed in less than 2 h. The
189 concentrate obtained by this methodology was demonstrated to be active suppressing 8
190 lipopolysaccharide (LPS)-stimulated cytokine expression, thus it might be considered a
191 promising starting material for further lunasin purification. Another efficient and cheap
192 alternative to plant peptide-purification or chemical synthesis is recombinant production of
193 lunasin by transgenic organisms.34 The obtained yield of recombinant production was 75 mg/l
194 and 210 mg/l in Escherichia coli and Clostridium thermocellum, respectively.34,35 Lunasin
195 was successfully expressed as recombinant his-tagged gene in E. coli and was found to have
196 identically biological activity than that demonstrated for synthetic lunasin.36 Recently, a
197 patent has been granted to prepare lunasin from plants through expressing a fusion protein
198 comprising lunasin and cleaving it from given protein.37
199 Lunasin is currently commercialized as a dietary supplement with different health
200 claims.38 LunasSoyTM (protein complex) and LunasinXP® (peptide extract) are two powder
201 supplements (Soy Labs®, LLC, USA) with beneficial properties on heart and skin health,
202 immunomodulatory, anti-inflammatory and anti-aging effects. A soy protein drink mix
203 (Carefast FSP100, Carefast Products, Inc.) enriched with LunasinXP® has been developed. As
204 indicated by the manufacturer, this beverage delivers 13 g of heart-healthy soy protein and 30
205 mg of soy isoflavones per serving. LunaRich® is a product commercialized by Reliv
206 International (USA) that provides (in one 125 mg capsule) the same quantity of lunasin found
207 in 25 g of high-quality soy protein, the daily amount identified by the Food and Drug
208 Administration (FDA) to help reduce the risk of heart disease. LunaSmartTM Blend
209 (LunaSmart, USA) is also a capsule supplement that contains 600 ng of lunasin by serving
210 (two capsules). Recently, a new product has been launched by Simplesa® (USA), known as
211 LunaCellTM and defined as a new generation of lunasin products because of the effective and
212 potent combination of this peptide and protease inhibitors, which together deliver bioavailable
213 lunasin to the body.
214 9
215 BIOAVAILABILITY OF LUNASIN
216 One of the most important characteristics of any food-derived compound with
217 demonstrated biological properties is its ability to remain intact and bioactive after absorption,
218 distribution and metabolism, termed as bioavailability. This term refers to the portion of the
219 ingested compound that reaches circulation. In vitro bioavailability studies have reported that
220 isolated lunasin, either synthetic or soybean-purified, is easily digested after 2 minutes
221 incubation with simulated gastric or intestinal fluids.39 However, lunasin survives, at least
222 partially, the attack of digestive enzymes when is present in crude protein extracts purified
223 from soybean, remaining 60% and 80% of initial lunasin after proteolysis by pepsin and
224 pancreatin for 120 min, respectively.39 Results from these studies suggested the protective
225 role that naturally-occurring protease inhibitors, such as Bowman-Birk inhibitor (BBI) or
226 Kunitz-trypsin inhibitor might exert against lunasin’s proteolysis by digestive enzymes.
227 Hernández-Ledesma et al. observed the protection exerted by BBI when lunasin present in
228 soybean-derived foods was subjected to a sequential digestion with pepsin and pancreatin,
229 suggesting the importance of the lunasin:BBI ratio on this protection.30 This hypothesis has
230 been confirmed by Cruz-Huerta and co-workers that described the protective effect of the
231 major isoinhibitor of the Bowman-Birk family (IBB1) on the in vitro digestion of synthetic
232 lunasin simulating physiological conditions.40 In absence of IBB1, lunasin is completely
233 hydrolyzed by the action of digestive enzymes whereas partial hydrolysis of lunasin is
234 observed when the isoinhibitor is present. Peptides released from lunasin during simulated
235 digestive process have been recently identified.41 These authors also studied the
236 bioavailability of lunasin and released fragments by using Caco-2 cell monolayers, indicating
237 that both lunasin and fragment 11RKQLQGVN18 were absorbed intact across the intestinal
238 epithelium through a mechanism involving paracellular passive diffusion. The role of Kunitz-
10
239 trypsin inhibitor on the stability of lunasin present in soybean products against pepsin-
240 pancreatin hydrolysis has also been recently demonstrated.42
241 In vivo bioavailability studies carried out in animals and humans have confirmed
242 above in vitro findings. Hsieh et al. demonstrated that lunasin was bioavailable when orally
243 ingested by mice and rats.43 Synthetic 3H-labeled lunasin was absorbed in CD-1 mice and
244 distributed in various tissues including lung, mammary gland, prostate, and brain. In addition,
245 these authors also reported that lunasin extracted from the blood and liver of lunasin-enriched
246 soy flour-fed rats was able to suppress foci formation as effectively as equimolar amount of
247 synthetic lunasin. A previous study had also found an intact and active form of this peptide in
248 blood and liver of rats fed soybean lunasin-enriched diets.44 Interestingly, detection of lunasin
249 in plasma of healthy male volunteers after consumption of soy protein has been reported.45 In
250 these studies, subjects were fed 50 g of soy protein for 5 days after a one-week washout
251 period. Thirty and sixty minutes after soy protein ingestion, blood samples were collected and
252 analyzed for lunasin that appeared at concentrations between 50.2 and 110.6 ng ml-1 of
253 plasma. This amount represents 4.5% absorption of lunasin from 50 g of soy protein.
254 Recently, a method for the direct isolation and quantification of lunasin from plasma using an
255 ion-exchange microspin column and a validated immunoassay protocol has been optimized.6
256 The promising bioavailability results as well as the development of new technologies to detect
257 and quantify lunasin content in human samples are important in supporting clinical trials
258 confirming the chemoprevention evidence for this peptide by cell culture and animal studies.
259
260 CANCER CHEMOPREVENTIVE AND THERAPEUTIC PROPERTIES OF
261 LUNASIN
262 The promising chemopreventive properties of peptide lunasin have been demonstrated
263 in both cell culture experiments and animal models. Cell culture has demonstrated that while 11
264 lunasin’s treatment does affect neither normal cells proliferation nor morphology, it is capable
265 to prevent mammalian cells transformation induced by chemical carcinogens, viral oncogene
266 E1A, and ras-oncogene.46-48 Thus, a significant reduction of foci formation induced by
267 chemical carcinogens 7,12-dimethylbenz[a]-anthracene (DMBA) and 3-methylcholanthrene
268 (MCA) in mouse C3H 10T1/2 cells was reported after lunasin treatment.46 This peptide was
269 also able to increase p21 protein level, and to suppress foci formation in NIH/3T3 cells stably
270 transfected cells with the viral oncogene E1A and ras-oncogene.47,48 Differences between the
271 effect of lunasin in non-tumorigenic and tumorigenic cell lines have also been described.
272 Galvez et al. confirmed the selective capacity of this peptide to act on tumorigenic prostate
273 RWPE-1 cancer cells without affecting non-tumorigenic RWPE-2 prostate epithelial cells.49
274 The study also demonstrated that HIF1A, PRKAR1A, TOB1, and THBS1 genes expression
275 was up-regulated by lunasin in RWPE-1 cells, but not in RWPE-2 cells. In addition, a recent
276 study has reported that treatment of highly purified soybean-derived lunasin caused inhibition
277 of cell-line specific proliferation on anchorage-dependent growth of non-small lung cancer
278 cells, whereas it did not exert any effect on two normal bronchial epithelial cell lines.50
279
280 Lunasin modulates cell growth and apoptosis
281 Disruption of the normal regulation of cell cycle progression and division are
282 important events in the development of cancer. Therefore, inhibition of deregulated cell cycle
283 progress in cancer cells is being considered an effective strategy to delay or halt tumor
284 growth. Cell cycle is highly regulated by internal checkpoints molecules, such as the protein
285 kinases cyclin-dependent complexes (CDKs) after binding to their related cyclin regulatory
286 subunits.51 A significant arrest of breast cancer MDA-MB-231 cell cycle was observed when
287 these cells were treated with lunasin. This effect was accompanied of a decrease in the
288 expression of CDC25A, caspase-8, Ets2, Myc, Erbb2, PIK3R1 and Jun genes52,53 as well as in 12
289 the protein levels of cyclin D1, D3, CDK4 and CDK6.54 The increase of retinoblastoma (Rb)
290 gene expression and the inhibition of Rb phosphorylation might also contribute to the breast
291 cancer cell cycle arresting effect.17,52 Recently, lunasin has been shown to inhibit cell cycle
292 progression at the G1/S phase through alteration of the expression of CDK complex
293 components cyclin D1, CDK4, and CDK6, increase of p27Kip1 levels, reduction of Akt
294 phosphorylation, and inhibition of Rb phosphorylation in non-small lung cancer cells.50 These
295 authors showed that the in vitro effects of lunasin on this type of cells was significantly higher
296 in anchorage-independent assays and correlate significantly with its in vivo effects in a non-
297 small lung cancer cells mouse xenograft model. Inaba and coworkers compared the effects of
298 lunasin treatment on lunasin-sensitive (H661) and lunasin-insensitive (H1299) non-small cell
299 lung cancer cells with respect to lunasin uptake, histone acetylation and integrin signaling.55
300 These authors reported changes in histone acetylation in both cell lines, with H661 cells
301 showing a unique increase in H4K16 acetylation. Moreover, they demonstrated that the
302 lunasin´s ability to block the G1/S phase transition in H661 cells was also due to its ability to
303 bind αv-containing integrins (αvβ3 and αvβ1), and consequently act as an integrin signaling
304 antagonist.55 Integrins are heterodimeric cell-surface proteins involved in cell adhesion to
305 extracellular matrix and cell signaling that affect cell migration, survival and proliferation.
306 Therefore, the dual mechanism of action demonstrated for lunasin on non-small cell lung
307 cancer cells may be responsible for the potential therapeutic effects attributed to this peptide.
308 In colon cancer cells, lunasin caused a cell cycle arrest at G2/M phase and a
309 mitochondrial-mediated apoptosis induction accompanied of an increase of p21 protein
310 expression in HT-29 cells, and an increase of both p21 and p27 proteins in KM12L4 cells.56,57
311 In addition, lunasin’s treatment to KM12L4 cells affected the Bax:Bcl-2 ratio by up-
312 regulating anti-apoptotic Bax protein and down-regulating pro-apoptotic Bcl-2 protein,
313 resulting in the cytochrome c release involved in the mitochondrial-dependent apoptosis 13
314 pathway.56 Lunasin has also been demonstrated to exert cytotoxic effect against L12120
315 leukemia cells through arresting cell cycle at G2/M phase and inducing apoptosis.58 These
316 authors found that the increase of apoptotic cell number was due to the augment of caspases -
317 3, -8 and -9 expression. In human MCF-7 breast cancer cells, treatment of lunasin induced
318 tumor suppressor phosphatase and tensin homolog (PTEN)-mediated apoptosis after
319 increasing the expression of this protein and enhancing its nuclear localization.59 Moreover,
320 lunasin can act as co-adjuvant in breast cancer chemoprevention strategies because of the
321 synergistic effects were observed on cell cycle arrest and apoptosis induction, when combined
322 with natural and synthetic compounds, such as anacardic acid and aspirin, respectively.52,53,60
323 The angiogenesis and metastasis abilities of cancer cells are often associated to
324 deregulation and degradation of the extracellular matrix, and alteration in the integrin
325 receptors.61,62 These receptors can recognize peptides with RGD-motif in their sequences that
326 bind integrins to block their signaling pathways.63 The presence of this motif within lunasin’s
327 sequence could be responsible for its cell growth suppressive effects. Preliminary results in
328 colon cancer KM12L4 cells have demonstrated that lunasin’s treatment modulates the
329 expression of 62 genes involved in cell adhesion and extracellular matrix.57 In addition,
330 decreases in gene expression of collagen type VII α1, integrin β2, matrix metalloproteinase
331 10, selectin E and integrin α5, and increases in collagen type XIV α1 genes expression were
332 observed in lunasin-treated colon cancer cells compared with untreated cells.64 Similar effects
333 have been reported in breast cancer cells lines in which lunasin treatment suppresses cell
334 migration and invasion by inhibiting matrix metalloproteinases.65 Moreover, this anti-
335 metastasis property of lunasin has also been demonstrated to modestly inhibit the
336 angiogenesis-mediator vascular endothelial growth factor (VEGF) in 4T1 breast cancer cell.66
337 These results suggested the ability of lunasin to act as an anti-metastatic agent contributing on
338 its chemopreventive effects. 14
339 Increasing evidence confirms that colorectal cancer (CRC) could be initiated due to
340 metabolic dysregulation. Several studies pointed out an association between colonic cell
341 proliferation and insulin secretion,67,68 which led to other investigations on the potential role
342 of the antidiabetic drug metmorfin as an anticancer agent in both diabetic and non diabetic
343 patients, and animal models.69-71 Results from these studies showed positive effects of
344 metmorfin on CRC incidence, colon epithelial cell proliferation and number of aberrant foci.
345 A recent study showed that lunasin enhances drug efficacy of metmorfin in an in vitro model
346 of aggressive colon cancer (HCT116) in a hyperinsulinemic environment.72 Molecular
347 mechanisms for these effects include down-regulation of the tumor suppressor PTEN and the
348 lipid biosynthesis-associated enzyme fatty acid synthase (FASN), further blocking the self-
349 renewal and expansion of colon-like stem cells epithelial subpopulations. These findings
350 suggest the potential value of lunasin inhibiting CRC progression and conceivably, metastasis
351 and recurrence.
352 Melanoma is a type of skin cancer which major cause of morbidity and mortality is
353 associated with recurrence of this disease. It is initiated and maintained by a small population
354 of malignant cells called cancer-initiating cells that exhibit stem-cell-like properties and are
355 resistant to chemotherapies and radiation, suggesting their implication in invasion, metastasis,
356 and ultimately relapse.73 A recent study has demonstrated that treatment with lunasin reduced
357 the size of a subpopulation of melanoma cells expressing aldehyde dehydrogenase, a cancer-
358 initiating cells marker, associated with a reduction in the capacity to form colonies in soft agar
359 assays.74 Moreover, the mechanistic model revealed that lunasin, added to isolated melanoma
360 cancer-initiating cells, induced expression of the melanocyte-associated differentiation
361 markers tyrosinase and microphthalmia-associated transcription factor concomitant with
362 reduced expression of the stem-cell-associated marker NANOG. A subsequent study of these
363 authors demonstrated robust antimetastatic properties of lunasin through alterating histone 15
364 acetylation patterns and modulating integrin signaling through FAK and PI3K/AKT
365 pathways.75 Lunasin is shown as a complex and multifaceted peptide with potential
366 therapeutic efficacy against malignant diseases in which recurrence resulting from cancer-
367 initiating cells is expected.
368
369 Lunasin exerts an epigenetic mechanism of action
370 Epigenetic changes (i.e., variation in phenotype or patterns of gene expression without
371 a change in the underlying DNA sequence) are crucial for the regulation and expression of
372 genes involved in the normal growth and development, and in the maintenance of cellular
373 function, but also, they play a key role in cancer initiation and development.76 Three distinct
374 mechanisms are well known to regulate the epigenome: DNA methylation, histone
375 modifications, and small-interfering RNAs. Although a number of histone modifications
376 certainly play important roles in epigenetic regulation, the acetylation has been clinically
377 associated with pathological epigenetic disruptions in cancer cells.77 In addition to its ability
378 to neutralize the charge of histones altering the chromatin structure and accessibility, histone
379 acetylation of lysine residues is a mechanism associated with binding of various
380 transcriptional activators and repressors.78 Under steady-state conditions, the core H3 and H4
381 histones are mostly in a deacetylated (repressed) state. Evidence has shown that after treating
382 mammalian cells with lunasin and histone deacetylation inhibitor sodium butyrate, lunasin
383 translocates to cellular nucleus and tightly binds to deacetylate core histones inhibiting
384 histone acetylation, which is perceived by the cell as an abnormal cell growth, resulting in
385 programmed cell death.46,48,79 Lunasin either synthetic,46,54 or extracted from natural seeds
386 16,17,24,44 has been shown to inhibit core histone H3 and H4 acetylation seeds. Moreover,
387 lunasin has demonstrated to compete with different histone acetyltransferases (HATs), such as
388 yGCN5 and PCAF, inhibiting the acetylation and repressing the cell cycle progression.44 On 16
389 the basis of these findings, lunasin has been suggested as the first natural peptide with an
390 epigenetic mechanism of action, although further studies should be carried out to completely
391 elucidate the nature of this mechanism.
392
393 In vivo cancer preventive properties of lunasin peptide
394 The promising chemopreventive role of lunasin has also been demonstrated in several
395 animal models. The first model using SENCAR mice showed that topical administration of
396 this peptide reduced chemical carcinogen-induced skin carcinoma incidence and delayed the
397 tumor development. A posterior carcinogen-induced mouse model also reported lunasin’s
398 ability to delay the appearance of papilloma by slowing epidermal cell proliferation.80 Studies
399 carried out in the last years have revealed that lunasin also exerts chemopreventive effects
400 against other types of cancer, such as breast, colon, and lung cancers. This peptide was found
401 to reduce the generation and incidence of chemical-induced mammary tumors in SENCAR
402 mice fed lunasin-enriched diet.81 A similar preventive effect was demonstrated in a chemical-
403 induced mammary tumor rat model after administration of an isoflavone-deprived soy peptide,
404 although the specific contribution of lunasin to the observed effects has not clarified yet.82
405 Intraperitoneal administration of lunasin has also been found to reduce tumor incidence,
406 generation and weight in a xenograft breast cancer MDA-MB-231 mouse model.43 The
407 analysis of histological tumor sections carried out by these authors also revealed the
408 capability of the peptide to inhibit cell proliferation through reducing Ki-67 biomarker, and
409 inducing cell death. Similarly, a decrease of proliferation cell nuclear antigen (PCNA) levels
410 in liver tumor and an increase of Bax:Bcl-2 ratio was observed after intraperitoneal
411 administration of lunasin to colorectal KM12L4 metastasis mice.57 These authors also showed
412 that oral administration of lunasin could significantly reduce the incidence of liver metastasis
413 in this animal model; however, no effects on PCNA levels were found.83 These discrepancies 17
414 suggest that the route of lunasin’s administration can be determinant on its chemopreventive
415 activity, thus further studies determining the route and optimal dose should be performed in
416 order to achieve the best effects.
417 A recent study has been reported the tumor growth and size reducing properties of
418 lunasin after its intraperitoneal administration to the xenograft lung cancer H1299 mice when
419 compared to untreated animals.50 In a melanoma xenograft mouse model, it has been shown
420 that the in vivo tumor growth initiated by the cancer-initiating cells population was
421 significantly impaired by treatment with lunasin.74 Also, when C57Bl/6 mice were subjected
422 to an experimental metastasis model of melanoma using B16-F10 cells, lunasin significantly
423 suppressed the ability of these cells to invade and proliferate in the lungs.75 Since several mice
424 in the lunasin-treated group did not display any macrometastasis sign, the authors suggested
425 the efficacy of lunasin to reduce or abolish metastatic burden. The lunasin’s ability to inhibit
426 subcutaneous tumor growth of murine models of melanoma and non-small cell lung cancer
427 has been also demonstrated.84
428
429 OTHER BIOACTIVITIES OF LUNASIN
430 In the past several years, accumulating evidence has shown that lunasin not only exerts
431 chemopreventive properties but also acts modulating inflammation and oxidative stress,
432 cardiovascular, central nervous, and immune systems (Figure 1). The detailed information of
433 these lunasin’s effects was described below in this section.
434
435 Antioxidant and anti-inflammatory activities
436 A growing body of evidence has revealed that chronic inflammation and/or oxidative
437 stress are involved in the development of cancer as well as different degenerative disorders,
438 including cardiovascular and neurodegenerative diseases.85,86 Therefore, antioxidant and anti- 18
439 inflammatory compounds are being considered as promising candidates to exert
440 chemopreventive effects against cancer and a number of diseases. Lunasin has demonstrated
441 to exert in vitro antioxidant activity through different mechanisms, including free radical and
442 iron scavenging capacity.87
443 Lunasin extracted from Solanum nigrum L. was showed to chelate ferrous ions, and
444 protect DNA from oxidative damage via blocking fenton reaction to suppress hydroxyl radical
445 generation.88 At cellular level, lunasin acts as a free radical scavenger and inhibitor of reactive
446 oxygen species production in murine RAW264.7 macrophages stimulated with LPS, and
447 intestinal epithelial Caco-2 cells challenged by chemicals hydrogen peroxide and tert-
448 butylhydroperoxide.87,89 Recently, the ability of lunasin to protect human liver HepG2 cells
449 from oxidative stress stimulated by tert-butylhydroperoxide through modulation of associated
450 biomarkers has been reported.90 Oxidative damage in the eye lens has been reported as a
451 major mechanism in the initiation and development of cataracts. Interestingly, Dai et al.
452 demonstrated that lunasin administration markedly inhibited the progression of d-galactose-
453 induced cataract in rats.91 The mechanisms of action responsible for lunasin´s protection from
454 lipid peroxidation were mediated through up-regulation of antioxidant enzymes and inhibition
455 of the activation of polyol pathway by decreasing aldose reductase activity.
456 Lunasin has also been demonstrated to exert anti-inflammatory properties. In LPS-
457 induced RAW264.7 cells, lunasin inhibited secretion of pro-inflammatory mediators
85 458 interleukine (IL)-6, tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2). Moreover,
459 inflammation-associated genes expression profile was affected by this peptide in LPS-treated
460 RAW264.7 cells.92 The possible mechanisms of these effects could involve the modulation of
461 cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nuclear factor (NF)-
462 κB pathways.93,94 Lunasin presents in natural quinoa has also been found to inhibit the
463 secretion of nitric oxide, TNF-α and IL-6 in RAW264.7 cells activated by LPS.95 Recently, it 19
464 has been revealed that lunasin acts as a potential anti-inflammatory agent not only in
465 macrophages but also in adipocytes, disrupting the crosstalk between these two cells,
466 suggested that auxiliary prevention or therapy in obesity-related inflammatory applications.96
467 Moreover, lunasin’s anti-inflammatory activity possibly benefits its chempreventive activity
468 in 4T1 breast cancer cell cultured in the condition of inflammatory adipocyte.66
469 The inhibition of NF-κB-dependent inflammatory mediators through down-regulation
470 of Akt phosphorylation and p65 protein expression was observed after treating human LPS-
471 challenged THP-1 macrophages with lunasin.97 Peptide interacted with αVβ3 integrin receptor
472 being internalized by cells where it exerts its anti-inflammatory effects.
473
474 Effects of lunasin on cardiovascular system
475 Cardiovascular disease is one of the most serious public health problems worldwide.
476 Daily consumption of 25 g of soy protein was approved by the Food and Drug Administration
477 as a health claim to reduce the risk of this disease.98 Lunasin, as one of the components of soy
478 protein, could contribute on this preventive effect due to its demonstrated activity decreasing
479 the biosynthesis of hepatic cholesterol in HepG2 cells by inhibiting 3-hydroxy-3-methyl-
480 glutaryl (HMG) CoA reductase gene expression, and augmenting cholesterol clearance by
481 increasing the low density lipoprotein (LDL) receptor levels.99,100 This hypocholesterolemic
482 effect was confirmed in LDL-receptor mutant pigs, whose LDL cholesterol levels decreased
483 after supplementation of their casein diets with lunasin-enriched soy extract.99 A posterior
484 study carried out with the same pig model has exposed that administration of a lunasin-
485 enriched food supplement in combination with a lunasin-enriched soy extract reduced free
486 fatty acid by increasing leptin and adiponectin levels in the plasma of the animals.100 A human
487 trial was designed to evaluate the effects of the previously reported supplement on blood
488 cholesterol, LDL, triglycerides and uric acid levels, revealing an important decrease after 60 20
489 days consumption of the supplement.101 Interestingly, these reducing effects were greater
490 among the diabetic and overweight individuals, suggesting lunasin´s ability for the control of
491 cardiovascular diseases associated with adjustment lipid imbalances.
492 Recently, in a nonalcoholic steatohepatitis model, C57BL/6 mice were fed a high fat
493 diet and CardioAid and lunasin was orally administered for 25 weeks. The serum of mice was
494 analyzed, the population of CD4+CD25+ lymphocytes and transforming growth factor beta
495 secretion were showed a significant increased, and the IL1- production was decreased.102 It
496 indicated that dietary treatment associated with blunting the liver damage resulting to improve
497 the histological nonalcoholic fatty liver disease activity score. Moreover, the biological
498 markers like serum triglyceride and glucose levels were observed in mice fed lunasin,
499 showing that lunasin exerts protective properties in mice fed the high fat diet.102 In addition,
500 the antioxidant and anti-inflammatory properties of lunasin could contribute on its potential
501 protective role against cardiovascular disorders. Specifically, lunasin has been shown to
502 interact with endocytosis pathways in human macrophages THP-1 which are associated with
503 integrin signaling, clathrin-coated vesicles and macropinosomes,103 thus contributing to the
504 control of cardiovascular disease.
505
506 Effects of lunasin on central nervous system
507 In animal studies, it was shown that lunasin, once orally ingested, is absorbed and
508 distributed through various tissues, included the brain after crossing blood-brain barrier.43,45
509 The central (motionless/neuroleptic/cataleptic) effects of this peptide have been recently
510 assessed by Dzirkale et al. that found local intracisternal administration of synthetic lunasin
511 into male C57BL/6 mice to cause a marked neuroleptic/cataleptic effect even at small doses
512 (0.1 nmol/mouse). Lunasin decreased the hyperlocomotor activity of amphetamine (a
513 dopamine releaser), and slightly affected apomorphine (a dopamine receptor agonist)-induced 21
514 climbing behavior.104 In contrast, lunasin did not affect ketamine (a N-methyl-D-Aspartate
515 (NMDA) receptor agonist) and bicuculline (an antagonist of GABAA receptor)-induced
516 effects. These findings confirmed that glutamate and GABAergic systems did not play an
517 essential role in lunasin’s central effects while affinity of this peptide for dopamine D1
518 receptors could have an important contribution. Further studies aimed to confirming this
519 hypothesis should be needed because there is a necessity for novel types of antipsychotic
520 drugs without serious side-effects.
521
522 Effects of lunasin on the immune system
523 Cytokine immunotherapy is a recent therapeutic strategy used to control the immune
524 surveillance to eliminate cancer cells, and to regulate imbalance immune responses. A recent
525 work has reported, by the first time, the immunomodulatory properties of peptide lunasin.105
526 These authors demonstrated robust synergistic effects of lunasin, and cytokines IL-2 and IL-
527 12 combination on increasing IFN-γ production and granzyme B expression by natural killer
528 cells in post-transplant lymphoma patients who are immune compromised. A simultaneous
529 study carried out with human dendritic cells showed that lunasin-treated cells not only
530 expressed elevated levels of co-stimulatory molecules CD86 and CD49, but also exhibited up-
531 regulation of IL-1, IL-6, and chemokines CCL3 and CCL4.106 Using a prophylactic mouse
532 model, these authors also demonstrated the adjuvant activity of lunasin when mixed with a
533 soluble antigen (ovalbumin) to protect animals against subsequent challenge. Therefore, the
534 results of the study suggest the promising application of lunasin as vaccine adjuvant inducing
535 dendritic cells maturation, which in turn improves the development of protective immune
536 responses to the vaccine antigens.
537 Rheumatoid arthritis is characterized by inflammatory changes of synovial tissues and
538 the formation of rheumatoid pannus. The first study carried out with synovial fibroblasts from 22
539 rheumatoid arthritis patients suggests the promising role of lunasin. After cells treatment with
540 this peptide, a significant decrease of IL-6, IL-8, and matrix metalloproteinase-3 productions
541 were observed, causing suppression of NF- B activation.107
542 Asthma is other immune hypersensitive disease associated with dysregulated T helper
543 type-II immunity leading to chronic eosinophilic inflammation in the airways. A recent study
544 conducted with an ovalbumin plus intranasal LPS-induced mouse model found that
545 intraperitoneal injection of peptide lunasin effectively suppressed allergic airway
546 inflammation through reduction of total cells, especially eosinophils, in bronchoalveolar
547 lavage fluid, and IL-4 secretion from mediastinal lymph nodes.108 In addition, an important
548 decrease of inflammatory Fizz1 gene expression and increase of regulatory T cells
549 accumulation were observed in the lunasin-treated mice group. All these recent findings
550 suggest an immunomodulatory role for lunasin through multiple mechanisms of action
551 making it as a promising agent to be potentially used in therapy of hypersensitive immune
552 disorders or as an adjuvant to enhance vaccination response. Human trials are needed to
553 confirm these effects to have an effective and safe application.
554
555 CONCLUDING REMARKS
556 After nineteen years from its discovery in soybean, lunasin has become one of the
557 most studied plant peptides because of its beneficial properties against chronic diseases. Cell
558 culture and animal models have demonstrated the chemopreventive role of lunasin against
559 skin, breast, colorectal cancers and lymphoma. In addition, studies carried out in the last years
560 have suggested a promising effect of this peptide against hypercholesterolemia, obesity,
561 metabolic syndrome and associated-cardiovascular diseases as well as protective effects in
562 inflammatory and immune regulated-diseases. Findings obtained from bioavailability and
563 bioactivity studies support the inclusion of lunasin-containing food products in the human diet. 23
564 However, clinical trials confirming the already evidential properties are limited. Genomics,
565 proteomics, epigenetics and metabolomics tools should be used as new disciplines to
566 elucidate the complete mechanism of action of this peptide. Other aspects, such as the search
567 of new lunasin’s sources, the optimization of techniques to enrich products with this peptide,
568 study of lunasin’s interactions with other food constituents affecting its activity and
569 bioavailability should be also designed to increase the value of this peptide and its preventive
570 and therapeutic potential against cancer, cardiovascular and immunological diseases.
571
572 Conflict of Interest
573 B.O. de Lumen is one of the co-inventors of patent on lunasin owned by the
574 University of California, Berkeley, and owner of the company Narra Biosciences that is
575 currently developing lunasin-based products.
576
577 Acknowledgements
578 C.-C. H. acknowledges funding from Ministry of Science and Technology, Taiwan
579 (MOST 103-2320-B-003-003-MY3). C. M.-V. and B. H.-L. acknowledge funding from
580 Ministry of Economy and Competitiveness (MINECO, Spain) (grants AGL2013-43247-R and
581 AGL2015-66886-R).
582
583
584
585
24
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37
Figure legend
Figure 1. Lunasin, a natural peptide, acts with multiple bioactivities, including cancer prevention, anti-oxidative, anti-inflammatory, cholesterol biosynthesis regulatory, and central nervous system and immune system modulatory activities.
38
Table 1. Methodology of quantification and peptide lunasin concentration (expressed as mg/g of protein and mg/100 g product) of different soybean-derived products
Lunasin concentration Type of food Quantification methodology Reference mg/g protein mg/100 g product Textured soybean LC/MS-TOF analyzer 20.9-22.5 1600.0-1800.0 32
Soybean flour LC/MS-TOF analyzer 14.0-17.1 700.0-800.0 32
Regular soymilk Western-Blot 4.7-5.6 11.8-15.7 30 ELISA 0.5-2.8 1.8-9.3 31 Organic soymilk Western-Blot 4.3-8.3 10.7-18.9 30 ELISA 1.0-2.7 2.9-9.1 31 Regular soy-based formula Western-Blot 7.3 4.1 30 ELISA 4.4-5.5 7.3-8.9 31 Organic soy-based formula Western-Blot 1.7-7.0 1.5-2.8 30 Soy shake ELISA 0.1-0.4 1.3-3.6 31 Regular tofu Western-Blot 40.7-104.8 3.5-14.3 30 Organic tofu Western-Blot 30.9 6.7 30 Processed tofu Western-Blot 48.3-77.3 0.4-5.5 30 Regular tempeh Western-Blot n.d. n.d. 30 Organic tempeh Western-Blot 24.3-39.7 6.1-8.2 30 Bean curd Western-Blot 0-165.8 0-9.5 30 Soybean curd Western-Blot 77.3-135.4 1.1-10.7 30 Su Jea Western-Blot n.d. n.d. 30 Natto Western-Blot n.d. n.d. 30 Miso Western-Blot n.d. n.d. 30 Soybean beverage powder Top-down mass spectrometry --- 22-23a 9 2.7-3.9b 9 a : Native lunasin b : Glycated lunasin n.d. not detected
39