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With
the advancement of basic and clinical medical research, the development of a variety of tumor conditions has been effectively delayed and controlled
.
However, cancer remains one of
the leading causes of death worldwide.
In recent years, the incidence of cancer and various chronic diseases has gradually increased
.
A large number of studies have confirmed that reasonable dietary intake can effectively prevent the occurrence of
cancer and chronic metabolic diseases.
Lunasin was first isolated from soybean as a functional active peptide, containing 44 amino acids with a variety of physiological activities
.
lunasin has been shown to improve obesity-induced inflammation caused by high-fat diet in C57BL/6J mice, regulate the body's immune response, and prevent immune and inflammation-mediated gastrointestinal disorders in humans
.
In addition, lunasin inhibited the formation of skin papillomas in mice, induced G2/M cycle arrest and apoptosis in HT-29 cells, and increased cysteine-aspartate protease 3 (caspase-3) activity by 77%.
The structure and amino acid sequence of a peptide play a decisive role
in its biological function.
The biological activity of Lunasin may be determined by RGD cell adhesion modules consisting of Arg, Gly, and Asp residues and by 9 aspartate residues located at the carboxyl terminus of lunasin
.
For example, lunasin's polyaspartate tail (poly-D) binds to deacetylated histones H3 and H4 in breast cancer cells, thereby influencing the formation of centromeromere complexes, inducing mitotic termination and apoptosis
in cancer cells.
However, the anti-cancer mechanism of lunasin still lacks systematic research
.
The development of omics technologies such as transcriptome and proteome has greatly promoted cancer-related scientific research
.
In order to fully understand the anti-cancer function and molecular mechanism of lunasin, Dr.
Hao Yuqiong and Dr.
Yang Xiushi, Chinese Academy of Agricultural Sciences, and Dr.
Guo Huimin, Shanghai Academy of Agricultural Sciences, took human breast cancer cells MDA-MB-231 as a model to systematically analyze the intracellular regulatory effect of lunasin intervention on MDA-MB-231 from the transcription level and protein level, and revealed the molecular mechanism
of lunasin inhibiting the proliferation of breast cancer cells.
Results and Discussion
Lunasin inhibits breast cancer cell proliferation
Using chemically synthesized lunasin polypeptide (purity >95%) as the research material, cell proliferation experiments showed that lunasin had a dose-effect relationship on the proliferation of MDA-MB-231 in breast cancer cells (Figure 1), and the half inhibitory concentration IC50 was about 149 μmol/L.
Figure 1 Lunasin inhibits the proliferation of breast cancer cells
Lunasin blocks cellular mitosis by inhibiting DNA replication and induces mitochondria-mediated endogenous apoptosis
Hierarchical clustering dendrogram, heat map, and volcano map analysis of transcriptome sequencing data (Figure 2) showed that the sequencing results were reliable, the test reproducibility was high, and there were significant differencesbetween different treatment groups.
Differentially expressed genes were screened for enrichment analysis by quantification and differential expression analysis of sample mRNA (Figure 3).
The GO results showed that bioregulation, cell complex organization or biogenesis, metabolic processes, cell proliferation, signaling, etc.
were significantly enriched.
The KEGG results showed that pyrimidine metabolism, cell cycle, DNA replication, P53 signaling and other pathways were significantly enriched
.
Further mining and analysis of differentially expressed genes showed that the expression of pro-apoptotic factors CYC1, APAF-1, BAK1, BAX genes and CASP 3, 7, 9 and 14 genes in the lunasin-treated group was significantly upregulated (Figure 3C
).
Lunasin intervention resulted in an increase in the BAX/BCL-2 ratio from 22.
9 to 210.
6, which led to increased mitochondrial outer membrane permeability, leading to the release of mitochondrial intermembrane proteins such as cytochrome C and interaction with Apaf-1, triggering apoptotic bodies to assemble, ultimately leading to caspase-mediated apoptosis
.
Real-time PCR analysis (Figure 4) further validates the reliability
of transcriptome analysis results.
Fig.
2 Clustering analysis of transcriptome data of DA-MB-231 cells, Fig.
3 Transcriptomic analysis of MDA-MB-231 cells, Fig.
4 Real-time PCR verification of key genes
Lunasin induces apoptosis via the lysosomal-mitochondrial axis
Proteome analysis showed that the lysosomal pathway was significantly enriched, the expression of lysosome-related proteins was significantly downregulated (Figure 5), the expression of membrane proteins LAMP1, LAMP2, CD63 and V-ATPase (ATP6V0A1) was inhibited, and the synthesis of a variety of proteolytic enzymes was blocked, including DNASE2, SMPD1, LIPA, ASAH1 and Cathepsin cathepsin family (CTSS, CTSL, CTSD, CTSH).。 There are more than 120 kinds of lysosomal membrane proteins, LAMP1 and LAMP2 are the most abundant membrane proteins, accounting for 80% of lysosomal membrane proteins, which is the most important protein to maintain the stability and integrity of lysosomal membranes, as a lysosomal marker, its expression level reflects the functional state
of lysosomes.
Lysosomal hydrolases, especially the Cathepsin family, are often used as indicators
of lysosomal function.
Lunasin peptide can significantly inhibit lysosomal metabolic pathway, and the expression of related proteins is significantly downregulated, which reduces the stability of lysosomal membranes and enhances permeability, which also suggests that lysosomal dysfunction
at this time.
Increased lysosomal membrane permeability is an important factor
in inducing apoptosis.
Transcriptome analysis has shown that lunasin can inhibit the proliferation
of cancer cells by inducing mitochondria-mediated endogenous apoptosis.
Proteome difference analysis found that lunasin intervention upregulated the expression of Bid, Apaf-1, Caspase 3 and 4 proteins, and downregulated the expression of anti-apoptotic protein Bcl-2, which further confirmed that lunasin inhibited the proliferation of MDA-MB-231 cells by regulating apoptosis, while lysosomes may participate in apoptosis in synergy with mitochondria
.
Fig.
5 Proteomic analysis
of MDA-MB-231 cells, Conclusion
Lunasin peptide can significantly inhibit the proliferation
of MDA-MB-231 in human breast cancer cells.
Based on the unique RGD cell adhesion motif and poly-D structure, lunasin can inhibit the expression of DNA replication-related genes and proteins through the lysosomal-mitochondrial axis, and highly activate caspase-mediated mitochondrial apoptosis, induce apoptosis, and exert anti-cancer functions
.
The first author
Hao Yuqiong, female, Ph.
D.
, is currently a postdoctoral fellow at the School of Life Sciences, Southern University of Science and Technology, and his main research direction is the development
of synthetic biology systems based on Bunsen tobacco.
Guo Huimin, female, Ph.
D.
, is currently an assistant researcher at the Institute of Biotechnology, Shanghai Academy of Agricultural Sciences, and his main research direction is the evaluation and development and utilization
of functional factors of mixed grains.
The corresponding author, Yang Xiushi, male, Ph.
D.
, is an assistant researcher at the Institute of Hemp Research, Chinese Academy of Agricultural Sciences,
and the secretary-general of
the Quinoa Professional Committee of the Chinese Crop Society.
His main research direction is the evaluation and utilization
of functional factors of plant origin.
He has presided over 9 projects such as sub-projects of the National Key R&D Program, Hebei Provincial Key R&D Program, and participated in 5 projects such as the National Key R&D Program, the Public Welfare Industry (Agriculture) Scientific Research Project, the National International Science and Technology Cooperation Project, and the Science and Technology Assistance Project for Developing Countries
.
He has published 57 papers and 23 SCI papers as the first or corresponding author
.
3 authorized patents, 2 industry standards, 3 scientific and technological achievements, 2 published translations, and 2 registered varieties.
Lunasin peptide promotes lysosome-mitochondrial mediated apoptosis and mitotic termination in MDA-MB-231 cells
Yuqiong Haoa,b,1, Huimin Guoc,1, Yechun Hongb, Xin Fana,Yumei Sud, Xiushi Yange,*, Guixing Rena
a Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
b Institute of Plant and Food Science, Department of Biology, Southern University of Science and Technology, Shenzhen 518055, China
c Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China
d College of Life Sciences, Xinjiang Agricultural University, Urumqi 830001, China
e Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China
1 These authors have contributed equally to this work.
*Corresponding author.
E-mail address: yangxiushi@caas.
cn
Abstract
Lunasin, a novel bioactive peptide, is well-known for its anti-proliferation activity.
However, the mechanism of this effect is still poorly reported.
Here, synthesized lunasin was used and its anti-proliferative function was observed at the concentration of 0.
25 mg/mL in human breast cancer cell MDA-MB-231.
Conjoint analysis of transcriptome and proteome of MDA-MB-231cells was further performed.
The results demonstrated that cysteinyl aspartate specific proteinase (CASP) 3, CASP 7, and CASP 14 were significantly up-regulated after lunasin exposure, together with an increased Bax/ Bcl-2 ratio from 22.
9 to 210.
6, which indicated that caspase-mediated mitochondria intrinsic apoptosis was highly activated.
Moreover, lysosomal pathway was significantly suppressed under lunasin exposure, suggesting that lysosome may cooperate with mitochondria to participate in apoptosis.
In addition, lunasin also down-regulated genes involved in DNA replication in MDA-MB-231 cells.
Overall, our study reveals that the anti-proliferation effect of lunasin peptide might be triggered via the inhibition of DNA replication and cell mitosis, as well as the promotion of lysosome-mitochondrial mediated cell apoptosis.
HAO Y Q, GUO H M, HONG Y C, et al.
Lunasin peptide promotes lysosome-mitochondrial mediated apoptosis and mitotic termination in MDA-MB-231 cells[J].
Food Science and Human Wellness, 2022, 11(6): 1598-1606.
DOI:10.
1016/j.
fshw.
2022.
06.
018.
