Overexpression of Soybean-Derived Lunasin in Wheat and Assessment of Its Anti-Proliferative Activity in Colorectal Cancer HT-29 Cells

Abstract

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 Agrobacterium-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 Agrobacterium-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.

Anti-Colon Cancer Activity of Novel Peptides Isolated from In Vitro Digestion of Quinoa Protein in Caco-2 Cells

“…Moreover, Choi et al have indicated that vorinostat can inhibit the expression of IL-6 and IL-8 in peripheral blood mononuclear cells (PBMCs) from treated patients [ 45 ]. In our previous study, lunasin of transgenic wheat also had inhibitory effect on colon cancer cells by modulating the apoptosis pathway, and the expression of Bax and caspase3 was increased while Bcl-2 was decreased [ 46 ]. Furthermore, TNF-α, VEGFA, and c-Myc were considered to serve important roles in tumorigenesis and tumor development, which can be regulated by HDAC1 [ 24 , 25 , 26 ].…”

Biological functions of peptides from legumes in gastrointestinal health. A review legume peptides with gastrointestinal protection

“…They proposed that Lunasin activated caspase‐3 and cleaved PARP could modulate the progression of the cell cycle progression mediated by the cyclin‐dependent kinase inhibitor p21. Similarly, Lunasin exerted antiproliferative activity by regulating the apoptosis pathway of HT‐29 human colon adenocarcinoma cells (Fan et al, 2020). On the other hand, González‐Montoya et al (2018) studied peptides from a soybean sprout protein isolate and found that fractions >10, 5–10, and <5 kDa caused cytotoxicity to Caco‐2, HT‐29, and HCT116 human colon cancer cells.…”

Application of enzymes in the preparation of wheat germ polypeptides and their biological activities

“…Lunasin was a unique 43-amino-acid cancer prevention peptide, which binded closely to deacetylated histone, destroying the kinetics of histone acetylation-deacetylation and leading to cell death, but this only occurred in transforming cells and does not affect normal cells. The polypeptide extracted from transgenic wheat had higher anticancer activity ( 81 – 83 ).…”