Lunasin peptide promotes lysosome-mitochondrial mediated apoptosis and mitotic termination in MDA-MB-231 cells

Yuqiong Ho, Huimin Guo, Yechun Hong, Xin Fn, Yumei Su, Xiushi Yng*, Guixing Ren

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

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.

Keywords:

Anti-proliferation

Transcriptome and proteome analysis

Cell apoptosis

DNA replication

1. Introduction

Improvements in basic and clinical cancer research have retarded the progression of lots of tumors. However, cancer remains one of the leading causes of mortality in the world. In flammation, as a part of the host deference system against external aggression and injury, is a critical component of tumor progression. Tumor microenvironment is largely depended on the orchestrating of inflammatory cells,which are crucial participant in the neoplastic process, fostering proliferation, survival, and migration. Moreover, tumor cells have shared some of the signal molecules of the innate immune system including selectins and chemokines for the invasion, migration, and metastasis [1]. Increasingly evidences supported that cancer and chronic disease can be prevented by modifications of nutritional and lifestyle habits. Dietary intervention was a major component of cancer and in flammation control. Adequate nutrition intake rich in bioactive components could significantly decrease the rate of cancer, such as soy iso flavones, quercetin, apigenin, anthocyanins, and lycopene [2-4].

Lunasin, a natural bioactive peptide with 44 amino acids,is a portion of soybean 2S albumin, plays an important role in promoting human health [5]. Accumulating evidences supported that lunasin peptide exhibited multiple biological functions including anti-inflammation, immunoregulatory, antioxidant, anti-cancer,and cholesterol-lowering activity [6-8]. Lunasin could ameliorate obesity-induced in flammation and regulate the immune responses in C57BL/6J mice fed a high-fat diet [9]. Peptide extracts from lunasin over-expressed transgenic rice showed enhanced antioxidant and anti-in flammation activity [10]. In addition, lunasin was characterized as an immunomodulatory peptide with the potential capacity to prevent immune and in flammatory-mediated disorders in the human gastrointestinal tract [11]. Lunasin exhibited strong anti-carcinogenic activity which has been demonstrated both by in vitro and in vivo assays. The formation of skin papilloma in mice was significantly blocked by lunasin without affecting the growth, proliferation, and morphology of normal cells [12]. Also, lunasin was reported to induce G2/M cell cycle arrest and apoptosis in HT-29 cells, increasing caspase-3 activity by 77% [13]. Hsieh et al. found that lunasin is actually the bioactive cancer preventive agent in soy Bowman-Birk inhibitor (BBI) concentrate, and BBI seemed to protect lunasin from digestion when soybean was ingested by humans [14].

Lunasin exhibited chemopreventive properties against breast cancer through modulating histone acetylation and protein expression [15].de Mejia et al. found that lunasin peptide inhibited inflammation in lipopolysaccharide (LPS)-induced RAW264.7 macrophage by suppressing nuclear factor kappa-B (NF-κB) pathway [6]. Each protein has its own unique structure and amino acid sequence that determines its particular functions. Sequence analysis showed that lunasin contained a RGD cell adhesion module that was responsible for its cell internalization and attachment to extracellular matrix.Besides, a structurally conserved helix region (KHIMEKIQ) for chromatin binding was found in its carboxyl terminal. Also, a polyaspartic acid tail (poly-D) bound to the deacetylated histone H3 and H4, thereby affecting the formation of centromere complex and resulting in mitotic arrest and cell death [16,17].

Advances in omic technology has greatly promoted the research of tumor formation, drug treatments, and nutrition healthcare.Omics studies combined with nutritional care could provide dietary guidance for human beings [18]. In a request for a comprehensive understanding of lunasin role in anti-cancer function and the possible molecular mechanism, here, lunasin peptide was synthesized and used for cell anti-proliferation assay by using human breast cancer cells MDA-MB-231. Conjoint analysis of transcriptome and proteome was conducted to comprehensively explore the intracellular regulation in MDA-MB-231 cells after co-cultivation with lunasin. Global change within the MDA-MB-231 cells under lunasin exposure at the transcript and protein levels was also explored to demonstrate the possible regulatory pathways of lunasin peptide in anti-proliferation effects.

2. Material and methods

2.1 Materials and reagents

Methanol, acetonitrile, formic acid (> 99%), phosphate-buffered saline (PBS), and protease inhibitor cocktail were purchased from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Lanbolide Biotechnology Co., Ltd. (Shanghai, China). Lunasin standard was synthesized by Bachem Company (Bubendorf,Switzerland). The primary rabbit polyclonal antibody against the 17 amino acid C-terminal peptide (EKHIMEKIQGRGDDDDD)was synthesized by Sangon Biotech Corporation (Shanghai, China).MDA-MB-231 cells were purchased from the Institute for Biological Science, Chinese Academy of Sciences (Shanghai, China).

2.2 Cell proliferation assay

Synthesized lunasin peptide was used as the standard in cell proliferation assay. Before the cell proliferation assay, the peptide feature was confirmed through ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)analysis coupled with Western blot [19]. Cell proliferation assay was carried out using the method of Zhu et al. [20] with minor modifications. MDA-MB-231 cells were cultured in DMEM medium (containing 10% FBS, 1% streptomycin, and 1% penicillin) at 37 °C in 5% CO2for the reproduction, and then were seeded in a 96-well plate with the concentration of 2.5 × 104cells/well. After 18 h incubation, the DMEM medium was removed and replaced with fresh DMEM medium containing a series of gradient concentrations of lunasin standard (0.062 5, 0.125, 0.25, 0.5, 1.0 mg/mL). Cell proliferation was determined after 72 h co-culture through the staining with Hanks Balanced Salt Solution (HBSS: 1.25% glutaraldehyde and 0.6% methylene blue). Absorbance was read at 570 nm using a microplate reader (Bio-Rad, Cambridge, MA, USA). Data was expressed as the percent of that from control group.

2.3 Transcriptomic analysis

Similar to the cell proliferation assay, MDA-MB-231 cells were seeded in a 96-well plate with the concentration of 2.5 × 104cells/well.After 72 h co-culture of MDA-MB-231 with synthetic lunasin, the DMEM medium was removed and replaced with 100 μL trizol for cell lysis and transcriptome analysis. MDA-MB-231 cells without lunasin treatment were identified as control group. In transcriptomic analysis,RNA was extracted and prepared for cDNA library construction. And then the libraries were sequenced using illumine Hiseq 4500 platform,generating the 150 bp pair-end raw reads. Raw data was analyzed for quality control. Clean reads were aligned to reference genome through HISAT2, and the fragments per kilobase per million (FPKM) of each gene was calculated based on the length of gene and reads count mapped to the gene. Differential expression analysis was performed using deseq package with quantile normalization methods, and false discovery rate (FDR) less than 0.05 was defined as differential expression. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes (DEGs) were carried out using the topGO package and KEGG Orthology-Based Annotation System (KOBAS), respectively. GO is an internationally genetic functional classification system, used for descripting the genes in different dimensions including biological process, cellular components, and molecular functions. KEGG is a database for systematically analyzing the metabolic pathways of gene products, facilitating to understand the gene functions. KOBAS is a tool for the annotation of sequences by KEGG Orthology terms.

2.4 Proteomic analysis

In proteomic analysis, total protein was ultrasonically extracted using lysis buffer (7 mol/L carbamide, 2 mol/L thiocarbamide, 0.1% CHAPS), and then centrifuged at 14 000 r/min for 20 min. After the isolation, the protein was digested with trypsin, and then labeled with regents according to the instruction of Tandem Mass Tag? (TMT)Kit (Thermo Fisher Scientific, Waltham, MA, USA). TMT is an in vitro peptide labeling technology, which enables simultaneous qualitative and quantitative analysis of proteins through using multiple isotope tags. In TMT 6-plex experiments, 6 samples including CK1,CK2, CK3, LUN1, LUN2, LUN3 were labeled with TMT 126, 127,128, 129, 130, and 131, respectively. For high pH fractionation, the labeled samples were reconstituted in 2% acetonitrile (pH 10.0),and then injected into a RIGOL L-3000 UPLC system for peptide separation through a reversed phase column (Durashell-C18,4.6 mm × 250 mm, 5 μm). Proteomic analysis was performed using the Orbitrap Fusion Lumos (Thermo Fisher Scientific),which connected a nanoflow reversed phase liquid chromatography (nanoRPLC). High pH fractions were collected and redissolved in 20 μL 12% methanol and 0.1% formic acid, and then analyzed using an Acclaim PepMap100 column (C18, 2 cm × 100 μm,5 μm) in the mobile phase with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The flow rate was 600 nL/min with the following steps: 0 min 7% B, 11 min 15% B, 48 min 25% B, 68 min 40% B, 69 min 100% B, and 75 min 100% B. Data were processed using Proteome Discoverer software 2.1 with the Uniprot_Human database.The identified proteins were normalized and analyzed for differential expression through t-test. The P value less than 0.05 (Foldchange > 1.2)was defined as differential expression. GO and KEGG enrichment analysis was also conducted based on the Uniprot_Human database.

2.5 Quantitative real-time PCR (qRT-PCR)

The qRT-PCR was performed on an ABI 7500 Fast Real-Time System (Applied Biosystems, Wakefield, WA, USA) to verify the accuracy of omics analysis as well as the expressions of key genes involved in DNA replication, cell mitosis and apoptosis pathways.The detail process was as follows: holding stage, 50 °C for 45 s,95 °C for 45 s; cycling stage, 95 °C for 15 s, 55 °C for 15 s, 72 °C for 45 s, 45 cycles; and melt curve stage, 95 °C for 15 s, 60 °C for 1 min, 95 °C for 30 s, 60 °C for 15 s. GAPDH was used as the internal reference gene to calculate the gene expression in qRT-PCR. Primer sequences can be found in the Table S1.

2.6 Statistical analysis

All the experiments above were conducted with three biological replicates. The data was expressed as the mean ± standard deviation,and was statistically analyzed through One-way analysis of variance(ANOVA), followed by Duncan’s multiple range tests using SPSS software. Statistical significance was set at P < 0.05.

3. Results and discussion

3.1 Anti-proliferation effect of synthetic lunasin on MDA-MB-231 cells

Anti-cancer activity of synthetic lunasin was measured by using human breast cancer cells MDA-MB-231 through cell proliferation assay. Before the cell proliferation assay, UPLC-MS/MS analysis of the synthetic lunasin was conducted to confirm the peptide feature. The chromatogram of lunasin standard through ESI system (Fig. S1) matched to the epitopes IQGRGDDDDDDDDD(783.2771++, Fig. S1A), KQLQGVNLTPCEK (505.6047+++,Fig. S1B), and WQHQQDSCR (415.5124+++, Fig. S1C). In the cell proliferation assay, as shown in Fig. 1, lunasin could inhibit the proliferation of human breast cancer MDA-MB-231 cells in a dose-dependent manner. Cell proliferation was significantly inhibited with the inhibition rates from 15.1% to 56.8% at concentrations from 0.25 mg/mL to 1.0 mg/mL, compared to the control group. In the previous study, lunasin extracts from soybean (lunasin content 0.53 mg/g) could also suppress the cell proliferation of MDA-MB-231 with an inhibition rate of 37% at the concentration of 2 mg/mL [21].Moreover, Hsieh et al. [22] also investigated the cell proliferation inhibitory effect of a synthesized lunasin peptide (purity > 95%)in MDA-MB-231 cells, and reported the IC50value of 181 μmol/L.In our study, the calculated IC50value of lunasin from a dose-effect trend line (not shown) was 149 μmol/L, which was slightly lower than that in the previous study. The tiny variation of IC50value might be due the different detection methods for cell proliferation, since the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)assay was used in the reported study. To further explore the global intracellular regulation in MDA-MB-231 cells, and reveal its anti-cancer function and possible molecular mechanism,MDA-MB-231 cells of the control group and 0.25 mg/mL lunasin group were collected to isolate the total RNA and protein for subsequent transcriptomic and proteomic analysis.

Fig. 1 Anti-proliferation assay of synthetic lunasin. The proliferation of MDA-MB-231 cells was significantly inhibited by lunasin standard. * and **indicate significant differences compared to the control group, P < 0.05 and P < 0.01, respectively.

3.2 Transcriptomic profiling in MDA-MB-231 cells

In transcriptomic analysis, there were average 65.14 million paired-end clean reads were generated after filtering out low-quality reads, and 96.2% of the reads were mapped to the reference genome (Table 1).Hierarchical cluster dendrogram and heatmap analysis showed a significant difference in the expression pattern between lunasin and control group (Figs. 2A, B). Setting a P < 0.05 and log2Foldchange > 1 or log2Foldchange < -1, differentially expressed genes (DEGs) were identified as up-regulated or down-regulated genes (Fig. 2C). To understand the differences in expression pattern between lunasin and control group, the DEGs were subjected to GO and KEGG enrichment analysis. The result of GO enrichment analysis was shown in Fig. 3A, DEGs were classified into three functional groups including biological process, cellular components, and molecular functions.The most abundant terms for biological process (BP)were “biological regulation”, “cellular component organization or biogenesis”, and “metabolic process”. Besides, “cell proliferation”and “signaling” were also significantly enriched. GO terms related to cellular components (CC) were enriched in “cell” and “organelle”.As for molecular functions (MF), the most highly represented GO term was “binding”, especially binding with DNA. KEGG enrichment analysis was shown in Fig. 3B, including pyrimidine metabolism,microRNAs in cancer, cell cycle, DNA replication, etc.

Fig. 2 Transcriptome profiling of MDA-MB-231 cells. (A) Hierarchical cluster dendrogram and (B) heatmap of normalized transcript abundances from 6 RNA-seq samples (CK and lunasin group, including three biological replicates). The cluster analysis showed a significant difference in the expression pattern between lunasin and control group. (C) Volcano plot of DEGs between lunasin and control group. Setting a P < 0.05 and log2 Foldchange > 1 or < -1, DEGs were identified as up-regulated (red) or down-regulated (green) genes.

Fig. 3 Transcriptomic analysis in MDA-MB-231 cells. (A) GO and (B) KEGG enrichment analysis of differentially expressed genes. DEGs were classified into 3 GO functional groups including BP, CC, and MF. Circle size and color in KEGG enrichment analysis indicated the gene counts and P value enriched in the pathway, respectively. (C) Cluster analysis of differentially expressed genes involved in cell apoptosis and mitosis (Foldchange after log2 transformation). Samples are displayed in columns and genes in rows, gene expression is represented as a color, with red for higher values and green for lower values. (D) Visualization of the genes involved in DNA replication (red boxes showed up regulation, green boxes showed down regulation).

Table 1Overview of the sequencing reads.

Similar to the results of GO and KEGG enrichment analysis, lunasin has been demonstrated to inhibit cell cycle progression at the G1/S phase in non-small lung cancer cells [23]. Additionally, lunasin could cause a cell cycle arrest at G2/M phase and a mitochondrial-mediated apoptosis in colon cancer cells [24,25]. DNA replication is a key part in the regulation of cell cycle. Deregulation of DNA replication and cell cycle underlies the aberrant cell proliferation, which are identified as the important events in the development of cancer. “MicroRNAs in cancer” was significantly enriched, which might be associated with the anti-cancer function of lunasin peptide. MicroRNAs are a group of endogenous small non-coding RNA molecules, regulating cell proliferation, cell cycle, cell death, cell apoptosis, etc. Some microRNAs are important mediators of p53 signaling pathway [26].p53 is a tumor suppressor protein, plays a central role in tumor suppression. The results of GO and KEGG enrichment analysis indicated the possible regulatory molecular mechanism of lunasin peptide in anti-proliferation effects, although it requires further analysis and validation.

3.3 Lunasin can induce cell apoptosis, inhibit DNA replication in MDA-MB-231

GO and KEGG enrichment analysis were further explored to reveal the detailed regulatory mechanism of lunasin in anti-proliferation function. As was shown in Fig. 3C, the transcript levels of a series of CASP-related genes includingCASP 3,7, and14were 4.29, 1.72 and 15.10-fold up-regulated, respectively, compared to the control group (Table S2).CASP 3, an executor of apoptosis,plays a crucial role in cell apoptosis. The results indicated that cell apoptosis might be activated in MDA-MB-231 cells under lunasin exposure. RGD motif together with integrin receptors constitutes a major recognition system for cell adhesion, which play important roles in regulating cell growth, differentiation, and apoptosis [27,28].RGD motif in lunasin peptide will possibly contribute to the attachment to extracellular matrix, and inducing cell apoptosis through a caspase-dependent pathway [29]. Moreover, lunasin was reported to induce the apoptosis of human colon cancer cells through the activation of mitochondrial pathway by increasing the expression of Bax, decreasing Bcl-2 expression and promoting the activation of CASP 3 [13]. Besides, lunasin exerted anti-proliferative activity through apoptosis pathway in colorectal cancer cells HT-29 via elevating the expression of p21, Bax, and CASP 3, and decreasing Bcl-2 expression [30]. In this study, the transcript levels of pro-apoptotic genes in MDA-MB-231 cells such as Bak1 (3.64-fold), Bax (1.4-fold),Cytochrome c (2.38-fold), and apoptotic peptidase activating factor 1(Apaf-1, 1.92-fold) were significantly enhanced after co-culture with lunasin (Fig. 3C and Table S2). Bcl-2 expression was highly decreased by 4.52-fold under lunasin exposure. Increased Bax/Bcl-2 ratio would stimulate mitochondrial outer membrane permeabilization (MOMP) and initiate the intrinsic apoptotic pathway. Following MOMP, mitochondrial intermembrane space proteins such as Smac and Cytochrome c are released into the cytosol. Cytochrome c interacts with Apaf-1, triggering apoptosome assembly, which eventually causes caspase-mediated apoptosis [31]. Here, Bax/Bcl-2 ratio was increased from 22.9 to 210.6,indicated that lunasin inhibited MDA-MB-231 cells proliferation via mitochondria-mediated intrinsic apoptotic pathway.

Genes involved in DNA replication were significantly downregulated under lunasin exposure (Fig. 3C and Table S2), especially the minichromosome maintenance (MCM) complex-related genes.Expressions of MCM 3, 4, 5, and 6 were 6.48, 2.81, 3.10, and 1.78-fold down-regulated, respectively (Fig. 3D). DNA unwinding is a key step in DNA replication, the core component of DNA helicase is MCM complex, taking part in the formation of replication forks and the recruitment of related proteins [32]. Besides, genes expression involved in DNA polymerase α-primase complex (POLA1,POLA2, PRIM1, and PRIM2) were 5.60, 4.81, 4.92, and 2.69-fold down-regulated after lunasin treatment. DNA polymerase α-primase complex, exhibiting DNA-directed 5’-3’ RNA polymerase activity,plays an important role in DNA replication initiation [33]. The results suggested that cell mitosis and DNA replication were highly inhibited by lunasin in MDA-MB-231 cells.

In qRT-PCR analysis, genes transcript level of CASP 3, 7,Bak1, Bax, and Cyc1 were significantly up-regulated, while Bcl2,MCM6 and POLA2 were highly down-regulated, compared to the control group (Fig. 4). The results further confirmed the reliability of transcriptomic analysis.

Fig. 4 Verification of qRT-PCR of key genes (Foldchange after log2 transformation).

3.4 Proteomic profiling in MDA-MB-231 cells

To explore the regulation of global protein levels in MDA-MB-231 cells under lunasin exposure, proteomic profiling of the cells (CK,Lunasin group) was carried out in this study. After differential expression analysis, a total of 1 237 proteins were obtained, including 545 up-regulated and 692 down-regulated proteins. Hierarchical cluster analysis of differentially expressed proteins (DEPs) indicated the significant difference in the levels of protein expression between lunasin and control group (Fig. 5A). GO enrichment analysis was shown in Fig. S2, DEPs could be assigned one or more GO terms, the most abundant terms related to BP were “signal transduction”,“immune system process”, “cell death” and “cell proliferation”.“Cytoplasm”, “nucleus” in CC and “binding” in MF were also significantly enriched. GO analysis of DEPs and DEGs showed the identity at some extent. KEGG enrichment analysis was performed to explore the detail metabolic regulatory pathways in lunasin anti-proliferation function. Top 20 KEGG pathways about up- and down-regulated DEPs were shown in Fig. 5B, especially the pathways including “l(fā)ysosome”, “ECM-receptor interaction”, “cell adhesion molecules”, “cell cycle”, “pathways in cancer”, and “DNA replication” were significantly suppressed.

Subsequent protein expression analysis showed that multiple proteins involved in DNA replication and cell mitosis were highly suppressed, including MCM2, MCM3, MCM4, MCM5, MCM6,MCM7 and RNASEH2B (Fig. 5E and Table S3). Formation of the related complexes in cell mitosis anaphase was inhibited, therefore,proteins expression of ANAPC1, ANAPC7 and ANAPC10 were significantly down-regulated. Similar to the transcriptomic analysis,protein expressions analysis also confirmed that lunasin could regulate DNA replication and block cell mitosis. Conjoint analysis of transcriptome and proteome showed that DNA replication in cancer cells was significantly suppressed after co-cultivation with lunasin peptide, while caspase-mediated mitochondria intrinsic apoptosis was highly activated (Figs. 3 and 5). In the previous study, poly-D domain in lunasin peptide may lead to mitotic termination and apoptosis through affecting the formation of centromere complex in chromatin region, according to the inhibition activity of poly-D on histone acetylation [34]. Balance of histone acetylation in chromatin is important for cell cycle control, which associated with cell apoptosis and tumor suppressing in carcinogenesis. Thus, inhibition of histone acetylation is a proposed epigenetic mechanism for lunasin cancer-preventive function [35,36].

3.5 Lunasin promotes apoptosis through lysosomemitochondrial axis

KEGG enrichment analysis of DEPs was further explored, the lysosome may participate in lunasin anti-proliferation function in MDA-MB-231 cells. As was shown in Figs. 5B2and C, the lysosomal pathway was significantly suppressed under lunasin exposure.Lysosome is rich in more than 60 kinds of hydrolases, participating in nutrients digestion and material recycle through autophagy [37].There are more than 120 kinds of membrane proteins in lysosome,among which LAMP1 and LAMP2 are the most abundant membrane proteins; CD63 mainly participates in preventing the membrane degradation by hydrolases; and V-ATPase cooperatively transports protons into lysosomal lumen, maintains its optimal pH (4.6) for the digestive function [38-40]. LAMP1, LAMP2, and CD63 were reported as the lysosome markers, played crucial roles in maintaining the stability and integrity of lysosomal membrane, whose expressions reflected the functional status of lysosomes [41]. Here, lysosomal membrane proteins including LAMP1, LAMP2, CD63 and V-ATPase were highly down-regulated at lunasin group, indicated the imbalance and increasing permeability of lysosomal membrane (Fig. 5D and Table S3). Lysosomal hydrolases, especially the cathepsin family,were often used as an indicator of lysosomal function [42,43]. As shown in Fig. 5D, expression levels of various hydrolases were significantly inhibited, including DNASE2, SMPD1, LIPA, ASAH1,and cathepsin family (CTSS, CTSL, CTSD, and CTSH). The results indicated the dysfunction of lysosome.

Fig. 5 Proteomic analysis in MDA-MB- 231 cells. (A) Hierarchical cluster analysis of DEPs. All the relevant proteins were grouped by hierarchical clustering based on expression values (log2 ratios) across all the samples. Samples are displayed in columns and proteins in rows. (B) KEGG enrichment analysis about up- (1)and down-regulated (2) DEPs. Circle size and color in KEGG enrichment analysis indicated the protein counts and P value enriched in the pathway, respectively.(C) Visualization of the proteins involved in lysosomal pathway (green boxes showed down regulation). Cluster analysis of DEPs involved in (D) lysosomal metabolic pathway and (E) cell mitosis. Samples are displayed in columns and proteins in rows. Protein expression is represented as a color, with red for higher values and green for lower values.

Increased membrane permeability of lysosomes would result in the release of hydrolases into cytoplasm, especially the cathepsin family, which was an important prerequisite to induce intrinsic apoptosis. Cathepsin B, H, and L could shear and activate the pro-apoptotic protein Bid and Bax, stimulate mitochondrial outer membrane permeabilization, promote the release of Cytochrome c,Apaf-1, and Smac into cytoplasm, andfinally induce caspase-mediated intrinsic apoptosis [44,45]. Studies have shown that cathepsin B could also directly activate caspase family and induce apoptosis [46,47]. In proteomic analysis, protein expression in MDA-MB-231 cells including Bid, Apaf-1, CASP 3, and CASP 4 was highly activated, whereas anti-apoptotic protein Bcl-2 was significantly inhibited after co-cultivation with lunasin (Fig. 5D).Proteomic analysis further demonstrated that lunasin peptide can inhibit the proliferation of MDA-MB-231 cells by regulating caspase-mediated intrinsic apoptosis. Moreover, lysosome may cooperate with mitochondria to participate in apoptosis.

4. Conclusion

Evidences about beneficial effects of lunasin peptide have promoted the nutritional supplements of lunasin to present in the market, while concerns on the functional mechanism of lunasin has been increasing. In this study, synthesized lunasin peptide significantly inhibited the proliferation of human breast cancer cells MDA-MB-231. Genes and proteins expression associated with DNA replication in MDA-MB-231 cells were significantly suppressed after lunasin treatment, and caspase-mediated mitochondria intrinsic apoptosis was highly activated. Lunasin peptide performed its cancer-preventive function through inhibiting DNA replication and inducing cell apoptosis via the lysosome-mitochondrial axis, probably on account of its RGD cell adhesion motif and poly-D structure.The results suggested a more comprehensive and safer theoretical evaluation system for the effective application of lunasin.

Con flicts of interest

The authors declare that they have no con flicts of interest.

Acknowledgment

The study wasfinancially supported by the Agricultural Science and Technology Innovation Program [CAAS-ASTIP-2021-ICS].

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://doi.org/10.1016/j.fshw.2022.06.018.