Scalable purification and characterization of the anticancer lunasin peptide from soybean.
ABSTRACT: Lunasin is a peptide derived from the soybean 2S albumin seed protein that has both anticancer and anti-inflammatory activities. Large-scale animal studies and human clinical trials to determine the efficacy of lunasin in vivo have been hampered by the cost of synthetic lunasin and the lack of a method for obtaining gram quantities of highly purified lunasin from plant sources. The goal of this study was to develop a large-scale method to generate highly purified lunasin from defatted soy flour. A scalable method was developed that utilizes the sequential application of anion-exchange chromatography, ultrafiltration, and reversed-phase chromatography. This method generates lunasin preparations of >99% purity with a yield of 442 mg/kg defatted soy flour. Mass spectrometry of the purified lunasin revealed that the peptide is 44 amino acids in length and represents the original published sequence of lunasin with an additional C-terminal asparagine residue. Histone-binding assays demonstrated that the biological activity of the purified lunasin was similar to that of synthetic lunasin. This study provides a robust method for purifying commercial-scale quantities of biologically-active lunasin and clearly identifies the predominant form of lunasin in soy flour. This method will greatly facilitate the development of lunasin as a potential nutraceutical or therapeutic anticancer agent.
Project description:Nutraceuticals have been proposed to exert positive effects on human health and confer protection against many chronic diseases. A major bioactive component of soy-based foods is lunasin peptide, which has potential to exert a major impact on the health of human consumers worldwide, but the biochemical features of dietary lunasin still remain poorly characterized. In this study, lunasin was purified from a soy-based food product via strong anion exchange solid phase extraction and then subjected to top-down mass spectrometry analysis that revealed in detail the molecular diversity of lunasin in processed soybean foods. We detected multiple glycated proteoforms together with potentially toxic advanced glycation end products (AGEs) derived from lunasin. In both cases, modification sites were Lys24 and Lys29 located at the helical region that shows structural homology with a conserved region of chromatin-binding proteins. The identified post-translational modifications may have an important repercussion on lunasin epigenetic regulatory capacity. Taking together, our results demonstrate the importance of proper chemical characterization of commercial processed food products to assess their impact on consumer's health and risk of chronic diseases.
Project description:<h4>Background</h4>The lower incidence of breast cancer among Asian women compared with Western countries has been partly attributed to soy in the Asian diet, leading to efforts to identify the bioactive components that are responsible. Soy Bowman Birk Inhibitor Concentrate (BBIC) is a known cancer preventive agent now in human clinical trials.<h4>Methodology/principal findings</h4>The objectives of this work are to establish the presence and delineate the in vitro activity of lunasin and BBI found in BBIC, and study their bioavailability after oral administration to mice and rats. We report that lunasin and BBI are the two main bioactive ingredients of BBIC based on inhibition of foci formation, lunasin being more efficacious than BBI on an equimolar basis. BBI and soy Kunitz Trypsin Inhibitor protect lunasin from in vitro digestion with pancreatin. Oral administration of (3)H-labeled lunasin with lunasin-enriched soy results in 30% of the peptide reaching target tissues in an intact and bioactive form. In a xenograft model of nude mice transplanted with human breast cancer MDA-MB-231 cells, intraperitoneal injections of lunasin, at 20 mg/kg and 4 mg/kg body weight, decrease tumor incidence by 49% and 33%, respectively, compared with the vehicle-treated group. In contrast, injection with BBI at 20 mg/kg body weight shows no effect on tumor incidence. Tumor generation is significantly reduced with the two doses of lunasin, while BBI is ineffective. Lunasin inhibits cell proliferation and induces cell death in the breast tumor sections.<h4>Conclusions/significance</h4>We conclude that lunasin is actually the bioactive cancer preventive agent in BBIC, and BBI simply protects lunasin from digestion when soybean and other seed foods are eaten by humans.
Project description:Lunasin, a bioactive peptide initially isolated from soybean, has anticancer, anti-inflammatory, and antioxidant activity. Due its great application value, lunasin seems to be a candidate gene in improving the nutritional value of crops. In this study, lunasin was inserted into the rice genome to evaluate whether it was feasible to express lunasin using the rice expression system and improve the bioactivity of protein in rice for our needs. We generatedlunasin-overexpressing rice lines, and chose three independent transgenic rice lines for further study. The lunasin content in trans-lunasin rice detected by UPLC-MS/MS was 1.01 × 10-3 g·kg-1 dry rice flour with grease removal in the lunasin extracts. The antioxidant efficacy of LET (lunasin-enriched fraction from trans-lunasin rice) and PEW (peptide-enriched fraction from wild type rice) was compared. Due to the presence of lunasin, LET showed higher (p < 0.05) antioxidant activity than PEW. LET exhibited high DPPH radical scavenging activity (IC50 value, 8 g·L-1), strong ABTS? radical scavenging activity (IC50 value, 1.18 g·L-1), and great oxygen radical scavenging activity (170 ?mol·L-1 Trolox equivalents when the concentration reached 4 g·L-1). Moreover, LET presented significantly higher (p < 0.05) anti-inflammatory activity on macrophage cells, and the NO production and the release of pro-inflammatory cytokines (IL-6, MCP1, and TNF-?) were significantly inhibited by LET. However, because of the low purity, LET showed weaker antioxidant and anti-inflammatory activity when compared to the Lunasin standard. These results suggested that it is feasible to use the rice expression system to express the exogenous lunasin in rice, and lunasin-overexpressing rice seems to be a candidate resource for application in functional food. Rice rich in lunasin is beneficial for human health, and could be used as a functional food in the diets of cancer and obese patients in the future.
Project description:Lunasin is a peptide from soybean seeds which has been demonstrated to have anticancer properties. It has also been reported in cereal seeds: wheat, rye, barley and Triticale. However, 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. This raises the question of the origin of the lunasin reported in cereal grain.
Project description:Alzheimer's disease (AD), a fatal progressive neurodegenerative disorder, also results from accumulation of amyloid-beta 42 (A?42) plaques. These A?42 plaques trigger oxidative stress, abnormal signaling, which results in neuronal death by unknown mechanism(s). We misexpress high levels of human A?42 in the differentiating retinal neurons of the Drosophila eye, which results in the Alzheimer's like neuropathology. Using our transgenic model, we tested a soy-derived protein Lunasin (Lun) for a possible role in rescuing neurodegeneration in retinal neurons. Lunasin is known to have anti-cancer effect and reduces stress and inflammation. We show that misexpression of Lunasin by transgenic approach can rescue A?42 mediated neurodegeneration by blocking cell death in retinal neurons, and results in restoration of axonal targeting from retina to brain. Misexpression of Lunasin downregulates the highly conserved cJun-N-terminal Kinase (JNK) signaling pathway. Activation of JNK signaling can prevent neuroprotective role of Lunasin in A?42 mediated neurodegeneration. This neuroprotective function of Lunasin is not dependent on retinal determination gene cascade in the Drosophila eye, and is independent of Wingless (Wg) and Decapentaplegic (Dpp) signaling pathways. Furthermore, Lunasin can significantly reduce mortality rate caused by misexpression of human A?42 in flies. Our studies identified the novel neuroprotective role of Lunasin peptide, a potential therapeutic agent that can ameliorate A?42 mediated neurodegeneration by downregulating JNK signaling.
Project description:The presence of lunasin was investigated in two soybean products, raw soy seeds and a commercial soybean beverage powder. Lunasin from the commercial soybean derivative was purified by reverse phase high pressure liquid chromatography and bystrong anion exchange solid phase extraction. Lunasin was further characterized by Top-down mass spectrometry (MS). The top-down characterization of lunasin unraveled a wide range of post-translationally modified proteoforms.
Project description:Lunasin is a 44 amino acid peptide with multiple functional domains including an aspartic acid tail, an RGD domain, and a chromatin-binding helical domain. We recently showed that Lunasin induced a phenotype switch of cancer initiating cells (CIC) out of the stem compartment by inducing melanocyte-associated differentiation markers while simultaneously reducing stem-cell-associated transcription factors. In the present study, we advance the hypothesis that Lunasin can reduce pools of melanoma cells with stem cell-like properties, and demonstrate that Lunasin treatment effectively inhibits the invasive potential of CICs in vitro as well as in vivo in a mouse experimental metastasis model. Mice receiving Lunasin treatment had significantly reduced pulmonary colonization after injection of highly metastatic B16-F10 melanoma cells compared to mice in the control group. Mechanistic studies demonstrate that Lunasin reduced activating phosphorylations of the intracellular kinases FAK and AKT as well as reduced histone acetylation of lysine residues in H3 and H4 histones. Using peptides with mutated activity domains, we functionally demonstrated that the RGD domain is necessary for Lunasin uptake and its ability to inhibit oncosphere formation by CICs, thus confirming that Lunasin's ability to affect CICs is at least in part due to the suppression of integrin signaling. Our studies suggest that Lunasin represents a unique anticancer agent that could be developed to help prevent metastasis and patient relapse by reducing the activity of CICs which are known to be resistant to current chemotherapies.
Project description:Lunasin is a soybean-derived peptide that exhibits anticancer bioactivity in different cancer cells and has been identified in different plants. However, recent studies revealed through molecular and chemical analyses that lunasin was absent in wheat and other cereals. In this study, the soybean-derived lunasin was cloned into pCAMBIA3300 and we transferred the expression vector into wheat via an <i>Agrobacterium</i>-mediated transformation. The identification of transgenic wheat was detected by polymerase chain reaction, Western blot analysis, and ultra-performance liquid chromatography with tandem mass spectrometry. An enzyme-linked immunosorbent assay showed that lunasin content in transgenic wheat L32-3, L32-6, and L33-1 was 308.63, 436.78, and 349.07 µg/g, respectively, while lunasin was not detected in wild-type wheat. Lunasin enrichment from transgenic wheat displayed an increased anti-proliferative activity compared with peptide enrichment from wild-type wheat in HT-29 cells. Moreover, the results of a real-time quantitative polymerase chain reaction showed a significant elevation in p21, Bax, and caspase-3 expression, while Bcl-2 was significantly downregulated. In conclusion, soybean-derived lunasin was successfully expressed in wheat via <i>Agrobacterium</i>-mediated transformation and may exert anti-proliferative activity by regulating the apoptosis pathway in HT-29 cells, which provides an effective approach to compensate for the absence of lunasin in wheat.
Project description:Lunasin is a naturally occurring peptide isolated from soybeans and has been explored in cancer treatment. Lunasin inhibits NF-?B activation and thus pro-inflammatory cytokine and mediator production in macrophages. In this study we demonstrate that lunasin can effectively suppress allergic airway inflammation in two murine models of asthma. In an OVA+Alum sensitization model, intranasal lunasin treatment at the time of OVA challenges significantly reduced total cells counts in bronchoalveolar lavage (BAL) fluid and eosinophilia, peribronchiolar inflammatory infiltration, goblet cell metaplasia and airway IL-4 production. In an OVA+LPS intranasal sensitization model, lunasin treatment either at the time of sensitization or challenge has similar effects in suppress allergic airway inflammation including significantly reduced total cell and eosinophil counts in BAL fluid, inflammatory gene Fizz1 expression in the lung, and IL-4 production by OVA re-stimulated cells from mediastinal lymph nodes. We further show that intranasal instillation of OVA+lunasin significantly increases OVA-specific regulatory T cell (Treg) accumulation in the lung comparing to OVA only treatment. Taken together, our results suggest lunasin as an anti-inflammatory agent can be potentially used in asthma therapy or as an adjuvant to enhance the induction of antigen-specific Tregs and thus boost the efficacy of allergy immunotherapy.
Project description:Numerous studies have identified certain components of soybean as having anticancer properties. Lunasin, a unique 43-amino acid soybean peptide, has been shown to suppress carcinogenesis in mammalian cells and mouse models. It has been hypothesized that lunasin modulates changes in chromatin organization by modifying histone tails, thereby, resulting in the expression of chemopreventive genes. In this report, microarray analysis was used to reveal a novel property of lunasin â?? its ability to up-regulate tumor suppressor and other chemopreventive genes in prostate epithelial cells. The effects of exogenous lunasin on the comprehensive gene expression profiles revealed that a total of 123 genes had a greater than twofold change in expression in the lunasin-treated cells as compared to the mock-treated cells. Of these genes, 121 genes were up-regulated in normal RWPE-1 cells and only two genes were up-regulated in malignant RWPE-2 epithelial cells. The genes up-regulated in RWPE-1 cells include those involved in tumor suppression, apoptosis, and the control of cell division. The results shed new light on lunasinâ??s previously observed chemopreventive properties. We propose that lunasin prevents cancer in vitro and in small animal models by up-regulating the expression of genes that prevent the onset of the disease. Experiment Overall Design: Four microarrays were analyzed. Two samples are RWPE1 human non-tumorigenic prostate epithelial cells and the other two samples are the genetically identical RWPE2 prostate epithelial cancer cells. In each cell line, one of the samples was treated with lunasin at 2 microM for 24 hr.