JPROT-01777; No of Pages 19 JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX

Available online at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/jprot

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Preventive and therapeutic potential of peptides from cereals against cancer☆

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Review

Margarita Ortiz-Martineza , Robert Winklerb , Silverio García-Laraa

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Center of Food Breeding, Tec de Monterrey, C.P. 64849 Monterrey, N.L., Mexico Dep. of Biotechnology and Biochemistry, CINVESTAV Unidad Irapuato, Irapuato Gto., Mexico

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AR TIC LE I NFO

ABSTR ACT

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Keywords:

Epidemiological studies have shown that regular consumption of food based on whole-grain

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Bioactive peptides

cereals and their products is associated with reduced risks of various types of degenerative 13

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Anticancer

chronic diseases. Food proteins are considered an important source of nutraceutical peptides 14

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Cereals

and amino acids that can exert biological functions to promote health and prevent disease, 15

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including cancer. There have been several reports on peptides with anti-tumour activity in 16

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recent years. Plant-derived peptides, such as rapeseed, amaranth and soybean lunasin have 17

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received main attention. In this review, we extend this vision to analyse the evidence of current 18 advances in peptides in cereals such as wheat, maize, rice, barley, rye and pseudocereals 19

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compared with soybean. We also show evidence of several mechanisms through which 20 bioactive peptide exerts anti-tumour activity. Finally, we report the current status of major 21

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strategies for the fractionation, isolation and characterisation of bioactive peptides in cereals.

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Biological significance

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In recent reports, it has been shown that peptides are an interesting alternative in the 25

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search for new treatments for cancer. One of the most studied sources of these peptides is

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Contents 1. 2.

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food proteins; however, a review that includes more recent findings for cereals as a potential source of bioactive peptides in the treatment of cancer, the techniques for their isolation and characterisation and the assays used to prove their bioactivity is not available. This review can be used as a tool in the search for new sources of anti-cancer peptides. The authors have no conflicts of interest, financial or otherwise. This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013. © 2014 Published by Elsevier B.V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peptide-based cancer therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

☆ This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013. E-mail address: [email protected] (S. García-Lara).

http://dx.doi.org/10.1016/j.jprot.2014.03.044 1874-3919/© 2014 Published by Elsevier B.V.

Please cite this article as: Ortiz-Martinez M, et al, Preventive and therapeutic potential of peptides from cereals against cancer, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044

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Epidemiological studies have shown that regular consumption of certain foods is associated with reduced risks of various types of chronic diseases, such as cardiovascular disease, type II diabetes, some types of cancer and major causes of mortality. One of the components of the diet reported to have the potential to prevent these diseases are whole grains and whole grain products. There are reports indicating that diets rich in whole grains or whole seed are associated with lower cancer mortality rates, particularly colon, breast and prostate cancers. This has led to more detailed studies on their disease-prevention activity and the potential therapeutic use of isolated components of food [1,2]. One of the most relevant groups of food derivatives with biological activity are proteins and peptide derivatives. Numerous studies have shown that food proteins are an important source of bioactive peptides. Those peptides are encrypted in the protein sequence, and once they are released, bioactive peptides exhibit several bio-functionalities and may have diverse therapeutic roles in human body systems. Abundant food-derived peptides exhibiting activities such as opiate, antithrombotic, anticancer, antihypertensive, immunomodulation, mineral-binding, antimicrobial or antioxidant properties have been reported [3,4]. Biologically active peptides are either naturally occurring or produced by enzymatic digestion or fermentation. Bowman– Birk-type and Kunitz-type trypsin inhibitors are examples of naturally occurring proteins with known nutraceutical functions, while products of protein enzymatic digestion or hydrolysates are the main sources of biologically active peptides in food-based research [5,6]. Sources of bioactive peptides are from animal origin and plant origin. Plant sources usually include cereals, such as wheat, corn, rice, barley, rye and pseudocereals, such as buckwheat and amaranth (Table 1). Other plant sources are legumes (soy, pea and chickpea), brassica species (mustard, rapeseed) and others

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(sunflower). Among plant sources, soybean is the most studied source of bioactive proteins and peptides. This can be explained by the fact that soybean is an important protein source, on the average, soybean contains about 40% protein [7,8]. Therefore, food proteins can be considered a source of nutraceutical peptides and amino acids which can exert biological functions to promote health and prevent disease, including cancer [5,6]. Proteins and peptides show potential results in preventing the different stages of cancer, including initiation, promotion and progression. Although there have been many reports on peptides with anti-tumour activity in recent years, these peptides are mainly derived from animals or microorganisms. Plant-derived peptides, such as rapeseed peptide, amaranth peptides and soybean lunasin have received great attention [9]. There are several mechanisms through which bioactive peptide exert anti-tumour activity, including:

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I. Induction of apoptosis; the process of apoptosis is carefully controlled, involving an energy-dependent cascade of molecular events led by cysteinyl aspartate-specific proteases called caspase. Strategies to overcome tumour resistance to either extrinsic or intrinsic apoptotic pathways includes activation of the extrinsic pathway through proapoptotic receptors, restoration of p53 activity, inhibition of the Bcl-2 family of proteins, BH3-only mimic proteins, caspase modulation, IAP inhibition and proteasome inhibition [10,11]. II. Blockage of intermediate tumour generation because its binding to cellular components related to cell proliferation and survival or biosynthetic pathways may modulate the growth rate of a tumour or even decrease its size [12,13]. III. Regulation of immune system may stimulate immunosurveillance by acting on cancer cells in several ways, for example by increasing the expression or presentation of tumour-associated antigens on the

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1. Introduction

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Plants as sources of bioactive peptides 3.1. Soybean . . . . . . . . . . . . . 3.1.1.Lunasin properties . . . . 3.2. Common bean . . . . . . . . . 4. Cereals source of bioactive peptides . . 4.1. Barley . . . . . . . . . . . . . . 4.2. Corn . . . . . . . . . . . . . . . 4.3. Oats . . . . . . . . . . . . . . . 4.4. Rice . . . . . . . . . . . . . . . 4.5. Rye . . . . . . . . . . . . . . . . 4.6. Triticale . . . . . . . . . . . . . 4.7. Pseudocereals (Amaranth) . . . 5. Peptide bio-characterisation . . . . . . 5.1. Isolation and fractionation . . . 5.1.1.Enzymatic hydrolysis . . . 5.1.2.Ultrafiltration . . . . . . . 5.1.3.Chromatographic methods 5.2. Characterisation of peptides . . 6. Conclusions . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . References . . . . . . . . . . . . . . . . . .

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JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX–XX X

Please cite this article as: Ortiz-Martinez M, et al, Preventive and therapeutic potential of peptides from cereals against cancer, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044

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Table 1 – Cereal sources of lunasin. Reference

Source

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Hyung J Jeong, Lam, and de Lumen (2002)

Barley

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H. Jeong and Jeong (2010)

Barley

Techniques Separation

Bioactivity assays Identification

N

Ion-exchange chromatography SDS-PAGE Western blot MALDI immunoaffinity column chromatography (matrix-assisted laser desorption ionisation) peptide mass mapping HPLC reverse phase HPLC (comparison with lunasin standard) Ion-exchange column chromatography Western blot (also for quantification)

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O

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t1:8

Nakurte et al. (2013)

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Hyung Jin Jeong et al. Rye (2009)

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Oats

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HPLC reverse phase coupled to an electrospray ionisation tandem mass spectrometer

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HPLC reverse phase

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HPLC (comparison with lunasin standard) Western blot

Histone acetylation assay: mouse fibroblast cells NIH 3T3 (ATCC) Colony assay: stable ras-transfected cells (NIH 3T3 cells), were used for the colony formation assay Bioavailability of lunasin from tissues of rats fed LEB (lunasin-enriched barley): four-wk-old male Sprague–Dawley rats fed LEB; the liver, kidney and blood were collected, and lunasin was isolated by ion-exchange column chromatography and purified by HPLC; lunasin was quantified by Western blot Inhibition assay of HAT activity: HAT Activity Colorimetric Assay kit (BioVision) Internalisation of barley lunasin: visualised by fluorescence microscopy after stain with antibodies labelled with fluorescent dye (NIH 3T3 cells) Inhibitory effect of lunasin on the cell cycle: determined the expression level of p21, p15INK4b, cyclin D1 and CDK4; NIH 3T3 cells by immunofluorescence stain Radical scavenging assay: DPPH radical scavenging assay Cell culturing: Human embryonic kidney HEK 293 (ATCC, catalogue no CRL-1573) Cell proliferation assay: MTT viability assay (HEK 293 cells) Bioavailability of lunasin from tissues of rats fed LER (lunasin-enriched rye): four-wk-old male Sprague–Dawley rats fed LER; the liver, kidney and blood were collected, and lunasin was isolated by ion-exchange column chromatography and purified by HPLC; lunasin was quantified by Western blot Inhibition assay of HAT activity: HAT Activity Colorimetric Assay kit (BioVision) Internalisation experiment: Immunostaining of 95% lunasin purified from rye and tissue lunasin internalised into the mouse fibroblast cell line NIH 3T3 Bioactivity assays are not reported

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Nakurte et al. (2012)

Triticale

HPLC reverse phase coupled to an electrospray ionisation tandem mass spectrometer

Pseudocereals Silva-Sánchez et al. (2008)

Amaranth Immunoprecipitation prior to identification assays

ELISA Western MALDI-TOF peptide mass mapping

JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX

Please cite this article as: Ortiz-Martinez M, et al, Preventive and therapeutic potential of peptides from cereals against cancer, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044

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Apoptosis and cell cycle distribution: The fraction containing the lunasin-like peptides was proved for their potential induction of apoptosis in HeLa cells; apoptosis assay was performed (Tunel Labeling Kits, RnDSystems) and cell cycle distribution using a FACS (fluorescence-activated cell sorting) apparatus; primary culture of fibroblasts was used as the control of normal cells

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In 2010, 50 peptide drugs were approved for marketing, mostly synthetic and recombinant hormone analogues, with annual global sales of around US$ 1 billion associated with the following peptide drugs: cyclosporine (e.g. Neoral®, Novartis), goserelin acetate (Zoladex ®, AstraZeneca), glatiramer acetate (Copaxone®, Teva Pharmaceuticals), leuprolide acetate (e.g. Lupron®, Abbott Laboratories) and octreotide acetate (Sandostatin®, Novartis). The increasing interest by the pharmaceutical industry in developing peptides as drugs is at least partially a consequence of the now widespread acceptance of protein therapeutics by physicians and patients and the development of solutions to problems such as a short half-life and molecule delivery [15,16].

2. Peptide-based cancer therapies

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Cancer remains a major source of mortality and morbidity around the world, despite numerous recent advances in treatment alternatives. Conventional cytotoxic therapies, such as radiation and chemotherapy, are the methods of choice for cancer management [17]. Chemotherapy is still the choice treatment for advanced and metastatic disease. However, both therapies have low therapeutic indices and are often highly toxic, with a broad spectrum of severe side effects. The development of a new class of anticancer that lack toxicity to healthy cells and are unaffected by common mechanisms of resistance would be a major advance in cancer chemotherapy [17,18]. Cancer cells provide their own growth signals to ignore growth inhibitory signals, avoid cell death, replicate without limit, sustain angiogenesis and invade tissues through basement membranes and capillary walls. In addition, the immune system fails to eliminate cancer cells due to the immunosuppressive effects mediated by tumour-infiltrating host cells. Cancer cells have an elevated apoptotic threshold, and the induction of apoptosis in cancer cells is increasingly seen as a therapeutic desirable goal [18,19]. Food proteins are considered not only nutrients for the proper maintenance of body functions but also as a source of important peptides with known biological activities. Food proteins can be considered a source of nutraceutical peptides that can exert biological functions to promote health and prevent disease, including cancer. Bioactive peptides have been known to be a part of the human diet for several years. With the appearance of chromatographic methods, the number of studies on bioactive peptides from animal and plant sources has increased. As the findings of these studies have shown, peptides exert regulatory functions besides their nutritional roles. Several studies have shown the anti-cancer potential of dietary proteins, peptides and amino acids, whether naturally occurring or the product of fermentation, enzymatic hydrolysis or gastrointestinal digestion, in the

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Initially, the search for bioactive peptides was mainly focussed on animal products such as milk. In the last years, the studies in plants were intensified, motivated by their huge diversity. Also nutritional studies with epidemiological basis have associated the consumption of certain foods with benefits for human health. Legumes are the plant source for which the most peptides with anticancer are reported. In the first instance this may be due to the high content of high quality proteins of legumes, but this first impulse has been fuelled by the discovery of proteins and peptides with interesting bioactivity, such as hemagglutinin, defensins and protease inhibitors.

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3.1. Soybean

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Legumes play an important role in a diet strategy for reducing cancer risk. Soybean (Glycine max) has undoubtedly received the most research attention, because it contains a variety of phytochemicals with demonstrated anti-cancer activity. The most widely studied bioactive substances are the isoflavones, the Bowman–Birk protease inhibitor (BBI) and the less purified BBI concentrate (BBIC). Soybeans also contains other proteins and peptides with biological activity, which may contribute to

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mediation of apoptosis and angiogenesis, important steps to control tumour metastasis [5,20]. Peptides have attracted attention as drug candidates owing to their possession of certain key advantages over alternative chemotherapy molecules. In contrast to most small-molecule drugs, peptides have high affinity, strong specificity and low toxicity, and in contrast to chemotherapeutic antibodies, they have good tissue penetration because of their small size. This provides impetus to the study of bioactive peptides as possible therapeutic agents, although the therapeutic use of peptides has remained limited due to their high instability in biological environments, rapid depuration from the blood, poor membrane transportability and effective digestion in the gastrointestinal tract. In vivo experiments and clinical trials are needed to demonstrate the physiological effect of peptides, but in vitro studies remain important prospective tools because peptide functionality is based on biological mechanisms. They cannot, however, replace in vivo and clinical studies because it is very difficult to establish a direct relationship between in vitro and in vivo biological activity. Peptide bioavailability after oral administration is one of the main reasons for this incomparability and one of the primary aspects to study before bioactive peptides can be incorporated into food or drug systems [18,21]. The implementation of a peptide-based therapy depends largely on its ability to remain intact until it reaches the target organ. Bioactive peptides must remain active and intact during gastrointestinal digestion and absorption to reach the cardiovascular system and potentially exercise their physiological effects, although once in the organism, all peptides must pass through a series of barriers that can inactivate them and consequently their biological action. This performance of anticancer peptides can be enhanced by using different delivery systems to improve stability and longevity, as well as to generate enhanced permeability and retention in the body (Table 2) [18,21].

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surface of cancer cells (antigenicity), by causing tumour cells to emit danger signals that stimulate innate or cognate immune responses by operating as adjuvants (immunogenicity) or by augmenting the propensity of tumour cells to be recognised and killed by immune effectors (susceptibility) [9,14].

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JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX–XX X

Please cite this article as: Ortiz-Martinez M, et al, Preventive and therapeutic potential of peptides from cereals against cancer, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044

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Lunasin is a 43-amino acid soy peptide that has been shown to be chemopreventive against oncogenes and chemical carcinogens in mammalian cells and in a skin cancer mouse model (Fig. 1). Soybean varieties display varying amounts of lunasin, which correlate with the extent of inhibition of core histone acetylation. Both, soy lunasin and synthetic lunasin inhibit core histone acetylation in a dose-dependent manner. Synthetic lunasin is heat-stable, resisting temperatures of up to 100 °C for 10 min. Animal studies indicate, that lunasin resists digestion and enters the target tissues after absorption [22,27]. During seed development, the lunasin peptide appears five weeks after flowering and persists in the mature seed. Western blot analysis of different soybean varieties and commercially available soy proteins shows the presence of the peptide in varying amounts. These results demonstrate the feasibility of producing large quantities of natural lunasin from soybean for animal and human studies. The high cost of synthesising lunasin makes it impractical to use synthetic lunasin for animal experiments and human studies. Therefore, there is a need to isolate, characterise and demonstrate the biological activity of lunasin [2]. Bioavailability studies carried out with animals have confirmed the preliminary results obtained by in vitro analysis. After the ingestion of lunasin-enriched soy and lunasin-enriched wheat by rats, lunasin was found as an intact and active peptide in the blood and liver of these animals. One of the properties of an ideal cancer-preventive agent is that it can be taken orally. This means being able to survive degradation by gastrointestinal and serum proteinases and peptidases and to reach the target organ or tissue in an active form. Simulation of the gastrointestinal digestion of lunasin has demonstrated that, while synthetic pure lunasin is easily hydrolysed by pepsin and pancreatin, lunasin in soy protein is resistant to the action of these enzymes. These results suggest that the combined protection provided by BBI and other naturally protease inhibitors, such as Kunitz trypsin inhibitor, against digestion plays a major role in making lunasin available in soy and wheat protein [12]. There have been several attempts to express the lunasin gene in E. coli. Sequence modifications should produce fusion peptides with desired characteristics [28,29]. Extensive searches of transcriptome and DNA sequence databases for wheat and other cereals have failed to identify sequences encoding either the lunasin peptide or a precursor protein, which leads to speculations about its real origin [30].

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Beans exert many effects beneficial to health, including the lowering blood glucose levels, blood lipids and incidence of cancer. Additionally, beans represent an inexpensive, but rich source of dietary proteins [31], whereas. Common bean (Phaseolus vulgaris L) accounts for 50% of legumes used for human consumption. Intensive investigations on various legume seeds revealed anti-tumour, anti-viral and antifungal activities [31–33]. Wang and Ng investigated a 7.3 kDa peptide from P. vulgaris cv. “Spotted bean” and which has considerable homology with defensins from other sources. The peptide displays potent antiproliferative activity for leukaemia cell line L1210 and lymphoma cell line MBL2 [34]. 2011, the isolation of a dimeric hemagglutinin was reported, which suppressed the proliferation of breast cancer MCF-7 cells (IC50 of 0.2 μM). The hemagglutinin-treated MCF-7 cells showed a number of changes, including cell cycle arrest in G2/M phase, phosphatidylserine externalisation and mitochondrial membrane depolarisation. The hemagglutinin induced apoptosis by activating the death receptor-mediated pathway, involving Fas ligands, caspase-8 activation, BID truncation, p53 release, caspase-9 activation and Lamin A/C truncation [31].

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3.2. Common bean

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the prevention and treatment of cancer [12,22,23]. The soybean Bowman–Birk protease inhibitor (BBI) is a 8 kDa polypeptide consisting of 71 amino acids and exhibiting two protease inhibitor sites, one for trypsin and one for chymotrypsin [24]. BBI works by inhibiting proteases involved in the initiation and promotion of carcinogenesis. Its capacity for preventing or suppressing carcinogenic processes has been demonstrated in different cell lines in vitro and in vivo [12,24]. The first reports of this proteases inhibitor are from the 1970s [25], but it is remarkable that the soybean was not considered as a potential source for other types of bioactive proteins or peptides until the 1980s, when a group reported the isolation of a polypeptide with an unusually high concentration of aspartic acid [26], later named lunasin.

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4. Cereals source of bioactive peptides

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Cereals can be defined as a grain or edible seed of the grass family, Gramineae (see Fig. 2). They are grown for their highly nutritious edible seeds, which are often referred to as grains. The grains consist of an embryo (or germ), the endosperm, which is packed with starch grains, and bran (fibre). If the cereal grain germinates, the seedling uses the nutrients provided by the endosperm until the development of a new plant occurs. Cereals are the most important sources of food, and cereal-based foods are a major source of energy from carbohydrates, protein, B vitamins and minerals for the world population [35]. Wheat, rice and corn are the major important grains in the human diet. The minor grains include oats, barley, rye, triticale, sorghum, millet and buckwheat. The cereals are protein-rich sources and therefore are a potential source of bioactive protein and peptides, already documented as imparting several physiological functions, including antioxidant, immunomodulatory, chemopreventive and anticancer functions [1,36]. (See Fig. 3.) The storage protein fractions of the cereal grains are categorised into four classes depending on their solubility: water-soluble albumins, globulins soluble in salt solution, prolamins soluble in alcohol solution and glutenins insoluble in neutral aqueous or saline solution and ethanol. The prolamins are monomeric polypeptide chains with molecular weights between 30 and 80 kDa. They are rich in proline and glutamine (20–55%). Prolamins in wheat are known as gliadins, in barley as hordeins, in rye as secalins and in oats as avenins [37].

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Barley (Hordeum vulgare L.) is the fourth most widely cultivated 367 cereal in the world after wheat (Triticum aestivum L.), rice 368

Please cite this article as: Ortiz-Martinez M, et al, Preventive and therapeutic potential of peptides from cereals against cancer, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044

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Table 2 – Mechanism of action of anticancer peptides from legumes and cereals. Reference Legumes Lam and Ng (2011)

Source

N

Treatment

Common bean (Phaseolus vulgaris cultivar “Legumi secchi”)

C

Isolated dimeric hemagglutinin with a relative molecular mass of 62000

O

Cell line or animal model

Mechanism

MCF-7 (human breast adenocarcinoma cell line)

Apoptosis through death receptor-mediated pathway

Evidence

R

R

E

Robles-Ramírez, Ramón-Gallegos, Mora-Escobedo, and Torres-Torres (2012)

Soybean (germinated)

Hydrolysate from soybean germinated for 6 days fraction >10 kDa

C

HeLa (human cervical adenocarcinoma cells) and HaCaT, non-cancerous human keratinocytes cell line

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Apoptosis

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Using a flow cytometer and differential staining they observe: • G2/M phase arrest • Phosphatidylserine externalisation • Mitochondrial membrane depolarisation DNA fragmentation was indiscernible by agarose electrophoresis Activation of death receptor-mediated pathway was detected by Western blot: Fas ligands aumented, caspase-8 activation, BID truncation, p53 release, caspase-9 activation and Lamin A/C truncation Apoptotic cells were identified through fluorescence microscopic observation of samples using Hoechst, staining revealed the typical changes, such as nuclear shrinkage, chromatin condensation and fragmentation. Images of phase contrast microscopy of HeLa cells treated with the peptide fraction show the membrane blebbing and cell shrinkage typical of apoptosis. The internucleosomal DNA fragmentation was determined by the Apoptotic DNA Ladder Kit that is based on Real-time quantitative PCR, the treated cells showed the characteristic DNA ladder pattern of apoptosis The caspase activity was evaluated using a fluorescence microscope after stain with a fluorescent kit for caspase 8 and 9 respectively, both caspase activity was found in the treated cells The PTTGl and TOP2A mRNA expression was determined by real-time quantitative PCR, the expression of both genes was markedly decreased by treatment. Analysis of cell cycle distribution was performed using flow cytometry after staining with propidium iodide shows that lunasin caused a G2/M cell cycle arrest on HT-29 colon cancer cells.

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Dia and Mejia (2010)

Soybean

Lunasin

HT-29 colon cancer cells

JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX–XX X

Please cite this article as: Ortiz-Martinez M, et al, Preventive and therapeutic potential of peptides from cereals against cancer, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044

t2:1

t2:8 Reference

Treatment

Mechanism

Cell line or animal model

Evidence

N

C

O

de Mejia, Wang, and Dia (2010)

Soybean

R

R

Hydrolysates Leukaemia cell rich on lunasin line L1210

Apoptosis through a caspase dependent pathway

E

C

T

E

D P

Cereals

Chen, Chen, Wu, Yu, and Liao (2010)

Rice (O. sativa L. Japonica)

Prolamin fraction

Human monoblastoid leukaemic cell line U937

R O

Potentiating of immune responses

The apoptotic status of the HT-29 colon cancer cells was confirmed by determining the presence of phosphatidylserine on the cell membrane using an Annexin V-FITC apoptosis detection kit by flow cytometry. Caspase-3 activity in HT-29 colon cancer cells was analysed using a fluorescence assay kit, the results were a statistically significant increase in caspase-3 activity The Bax protein expression increases upon treatment of lunasin. On the other hand, the expression of the anti-apoptotic Bcl-2 protein was dose-dependently reduced by lunasin treatment. Lunasin caused a dose-dependent increase in the expression of p21. This determination was done by Western blot Analysis of the cell cycle was performed by flow cytometry, treatment of L1210 leukaemia cells with LES for 24 h led to an increase in the amount of cells in the sub-G1 fraction in a dose-dependent manner. The apoptotic inducing effect was confirmed by microscopic analysis of the cells treated and stained with Hoechst reagent. Using a kit based on fluorescence the expressions of caspases 3, 8 and 9 were determined, treatment increased the expression of caspases 8 and 9 in concentration-dependent manner but mostly increased the expression of caspase-3 Analysis of p21 and p27 expression was performed by western blot, treatment showed no effect on the expression of p21 and p27. Medium supplemented with prolamin promoted monocyte differentiation of U937 cells, cell morphology was evaluated by cytocentrifugation onto a microscope slide stained with Wright's stain and observed under an inverted microscope for determining monocyte differential counts. The amount of TNF-α secreted significantly increased with prolamin treatment measured by enzyme- linked immunoassay (EIA).

JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX

Please cite this article as: Ortiz-Martinez M, et al, Preventive and therapeutic potential of peptides from cereals against cancer, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044

U

t2:8

t2:9

Source

O

F

(continued on next page)

7

8

t2:12 t2:10

N

Table 2 (continued) Reference Li et al. (2013)

C

Source

Treatment

Cell line or animal model

O

Corn Gluten meal (Zea mays)

R

Mechanism

Hydrolysate HepG2 (Human molecular hepatoma cell weight < 5 kDa line HepG2)

Apoptosis

Evidence

R

E

C

T

E

D

BALB/c mice transplanted with Mouse hepatoma 22 ascitic tumour (H22)

P

R O

Potentiating of immune responses

Microscopical observations revealed that the treatment affects the cell morphology and nuclear condensation and fragmentation appear in a dose-dependent manner. Cell-cycle phase distribution was analysed by flow cytometry with PI staining, the S phase cells increased, meanwhile the G0/G1 phase cells were markedly decreased. The expressions of several critical apoptosis related protein were checked by western blot analysis. Anti-apoptotic Bcl-2 expression was significantly inhibited in a dose-dependent manner, whereas that in Bax was relatively constant. The level of p53 was significantly increased. Simultaneously, the expression of Cleaved-caspase-3 was increased. The treatment could stimulate the growth and development of the thymus gland and spleen in H22-bearing mice Effect of CPs on IL-2 and TNF-a level in murine serum were determined by ELISA, the levels of IL-2, IFN-c and TNF-a were restored and enhanced in a dose- dependent manner

O

F

JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX–XX X

Please cite this article as: Ortiz-Martinez M, et al, Preventive and therapeutic potential of peptides from cereals against cancer, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044

t2:11

U

9

E

D

P

R O

O

F

JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX

374 375 376 377 378 379 380 381 382 383 384 385

E

373

R

372

R

371

(Oryza sativa L.) and corn (Zea mays L.). The low lysine content in the barley storage proteins limits their wide application as a nutritional ingredient in foods, so they are normally sold cheaply as animal feed [38]. Hordein, a barley prolamin, is the major protein in barley by-products, and it is the main storage barley protein. It is enriched with Glu, Pro, Leu, Val, Phe and Tyr, most of which have been reported to be related to antioxidant activity in their free forms or as residues in proteins and peptides. Hordein is composed of three sub-fractions, B hordein (sulphur-rich), C hordein (sulphur-poor), and D hordein (high molecular weight). C hordein has been reported as the fraction with higher antioxidant potency. Although limited recent data indicates that the antioxidant activity of barley hordein can be enhanced after enzymatic hydrolysis, there is little information regarding the effects of the type of protease and the hydrolysis process on the peptide structures and their antioxidant activity [38,39].

N C O

370

U

369

C

T

Fig. 1 – Taxonomy of the Gramineae Family. (Modified of McKevith, 2004 and Cavazos & Mejia, 2013).

In 2002, a peptide similar to lunasin was reported in barley, a cereal seed. Partially purified lunasin showed in vitro and in vivo bioactivity. This discovery motivated the search for lunasin in other cereal crops [40]. Currently, lunasin was found in seed crops such as soybean, amaranth, solanum family, wheat and rye. The presence of other bioactive components aside from peptides similar to lunasin is currently being investigated in these crops [41]. Nevertheless, sometimes the correct identification of lunasin is questioned, due to the use of methodology with low selectivity. [30,42].

386

4.2. Corn

396

Originating in the highlands of Mexico between 5000 and 10,000 years ago, maize (Zea mays L.) has become the most extensively cultivated cereal crop, followed by wheat and rice. Corn is an important source of protein. Globally, it contributes

397

Fig. 2 – Lunasin sequence. Please cite this article as: Ortiz-Martinez M, et al, Preventive and therapeutic potential of peptides from cereals against cancer, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044

387 388 389 390 391 392 393 394 395

398 399 400

10

R

R

E

C

T

E

D

P

R O

O

F

JOUR NAL OF P ROTEOM ICS XX ( 2014) X XX–XX X

404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419

C

403

approximately 42 million tons of protein a year, which corresponds to approximately 15% of the world's annual production of food-crop protein [43,44]. Maize has a wide range of food and non-food applications, and even when they are predominantly composed of carbohydrates, they contain considerable amounts of protein. The chemical composition of the grain is variable in different parts of the grain, with higher concentrations of protein in the endosperm (74%) and the germ (26%). The proteins of maize can be grouped according to their solubility as follows: albumins (water-soluble), globulins (saline-soluble), prolamins (soluble in strong alcohol solution) and glutelin (soluble in alkaline medium). The prolamin fraction (α-zein) represents the highest concentration in maize, representing 50 to 60% of the total protein [45]. Several types of corn peptides have been reported to have biological activity. In the search for peptides with inhibitory effects for the angiotensin-converting enzyme (ACE), the method of choice according to the reports is enzymatic hydrolysis, linked to separation using ultrafiltration membranes. Corn

N

402

U

401

O

Fig. 3 – Diagram with the most common strategies to produce and analyse bioactive peptides.

gluten meal (CGM), a major by-product of corn wet milling, contains 67–71% protein (w/w). At present, CGM is mainly used as forage. However, it may be considered a good source for the preparation of ACE inhibitor and antioxidant peptides because of its high proportion of hydrophobic amino acid and proline [46–49]. Corn gluten meal (CGM), a by-product of the starch industry with abundant protein, is mainly comprised of zein and glutelin. Corn protein is isolated from CGM, and in turn, corn peptides (CPs) are obtained from hydrolysis of the corn protein. Previous studies have found that CPs exhibited anti-breast cancer activity [50]; however, they ignored the effects of CPs on cancer cells and their underlying mechanisms. Some factors, such as molecular weight, hydrolysate concentration, degree of hydrolysis (DH) and amino acid composition, affects their activity [9,46–49]. More recently, a study was aimed at evaluating the antitumour mechanism of corn peptides (CPs). In vitro, the results showed that CPs significantly inhibited cell viability in both a dose- and a time-dependent manner. CP treatment induced S

Please cite this article as: Ortiz-Martinez M, et al, Preventive and therapeutic potential of peptides from cereals against cancer, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.03.044

420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438

11

JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX

4.4. Rice

469

Rice is not only an important cereal as a staple food worldwide but is also nutritional for human health, with fewer allergenic properties and easier digestion. Several ingredients isolated and derived from rice possess pharmacological and biological activities. Rice seeds contain about 8–9% protein. Four important fractions of rice proteins are identifiable by their differential solvent solubility. Of those, rice seeds contain 5–10% alcohol-soluble proteins (prolamin), 4–10% salt-soluble proteins (globulin and albumin) and 80–90% alkali soluble proteins (glutelin). The portion prolamin has proven to have a beneficial effect on activating anti-leukaemia immunity [53]. Rice protein isolate (RPI) has been reported to reduce the incidence of 7, 12-dimethylbenz[a]-anthracene-induced mammary tumours in rats. The potential role of phytochemicals associated with the RPI has been studied, but not the activity of the proteins and peptides, which are the main components [54]. Rice bran is a cheap co-product of rough rice milling, and it contains nutrients including B vitamins, minerals and fibre, including oil, which has health benefits. It is used as a low-cost animal feed. Defatting the bran, and directly hydrolysing the high-quality protein using endoprotease can sustainably release peptides in a consistent manner. The proteins in rice bran are complexed within carbohydrates and lipids and hence provide difficulties for protein extraction. Therefore, the direct hydrolysis of heat-stabilised defatted rice bran (HDRB) was performed to obtain high-quality and high-yield peptides for

452 453 454

458 459 460 461 462 463 464 465 466

470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494

C

451

E

450

R

449

R

448

N C O

447

U

446

4.5. Rye

514

F

468

445

O

467

Oat (Avena sativa L) is distinct among the cereals due to its multifunctional characteristics and its nutritional profile. Oat and oat by-products are used as complementary treatments for patients with diabetes and cardiovascular diseases. Recently, the ingestion of oat bran in a meal has been shown to affect gene sets associated with insulin secretion and b-cell development, protein synthesis and genes related to cancer diseases [51,52]. Oats also contain peptides similar to lunasin. Monitoring lunasin levels in different oat genotypes showed genotype-related variations over time. The results of antioxidant assays indicated that this oat lunasin-like peptide is bioactive [51].

444

R O

457

443

495

P

4.3. Oats

442

determining anti-cancer activities. This approach not only was unique but also can prove to be an economical way of producing anti-cancer peptides from rice bran on a large scale. Similar studies were able to obtain bioactive peptides from HDRB and to prove that the products of their hydrolysis with digestive enzymes retain and even improve their anti-cancer activity, but this information must also be tested in vivo [55]. In 2010, findings were published showing that the

Preventive and therapeutic potential of peptides from cereals against cancer.

Epidemiological studies have shown that regular consumption of food based on whole-grain cereals and their products is associated with reduced risks o...
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