VIT was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (111554, NICPBP, Beijing, China). TNBC cell lines, MDA-MB-231 and HCC-1937, were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). All cell lines were validated by STR profiling and tested negative for mycoplasma (C0298S, Beyotime, Shanghai, China). Cells were all cultured in a humidified incubator at 37 °C and 5% CO${}_2$. DMEM, RPMI-1640, and FBS were purchased from Gibco (12800082, 31800089, 16140071, Gibco, New York, USA). Specific antibodies for $\beta$-actin, HSP90AA1, CREBBP, and HIF-1A were purchased from the Proteintech group (20536-1-AP, 22277-1-AP, 20960-1-AP, Proteintech, Wuhan, China). Primary Antibody of HSP90AA1 was purchased from Beyotime (AF7140, Beyotime, Shanghai, China). Chemicals, such as SDS, and glycine for Western immunoblots were acquired from Beyotime Co, Ltd. (ST626, ST085, Beyotime, Shanghai, China).

The VIT targets were predicted using the online Super-PRED server (https://prediction.charite.de/subpages/target_prediction.php). Subsequently, we searched for these candidate targets in the UniProt database (https://www.uniprot.org) to identify which proteins interacted with VIT.

We collected TNBC-related genes from DisGeNET (https://www.disgenet.org/) and the Comparative Toxicogenomics database (http://ctdbase.org/) by using the keyword: “triple-negative breast cancer”. After removing repetitive targets, we obtained a list of insomnia targets. We identified the overlapping genes for VIT and TNBC using Veeny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/).

To visualize the relationship between VIT and TNBC, we used Cytoscape software 3.6 (San Diego, CA, USA) to create a network diagram based on the 37 overlapping genes obtained from integrating the potential targets of VIT with the TNBC-related genes.

To explore the impact of VIT on TNBC treatment, we performed GO and KEGG pathway enrichment analyses using the DAVID database (https://david.ncifcrf.gov/). The results of the GO and KEGG pathway enrichment analyses were visualized using R software 4. 2. 1 (R&R Software Co. Ltd., Budapest 1038, Hungary).

To investigate the interaction of the overlapping targets of VIT, a PPI network was generated using the STRING database (https://string-db.org/). The minimum interaction score required was set at $\ge$0.4, using the default parameters. The resulting PPI network was visualized using Cytoscape software 3.6 (San Diego, CA, USA).

The VIT 3D structure (No: 5280441) was from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and inputted into Open Babel software 3.1.1 (Pittsburgh, USA) to generate the mol2 formula. The HSP90AA1 (5H22), HIF-1A (3KCX), and CREBBP (4TQN) crystal structures were obtained from Protein Data Bank (https://www.rcsb.org). Subsequently, the water and ligand molecules were removed from the 3D structures using Discovery Studio 4.5 (Omaha, Nebraska, USA). The molecular docking analysis of VIT and its potential targets was performed using Discovery Studio to predict their interaction activity.

Survival information of the VIT hub genes following TNBC treatment was obtained by the Kaplan–Meier plotter (http://kmplot.com/analysis/), which was established based on the microarray and RNA-seq datasets for TNBC from TGCA [9].

MDA-MB-231 cells were cultured in DMEM, while HCC-1937 were cultured in RPMI-1640. Both these cell lines were supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO${}_2$.

MDA-MB-231 and HCC-1937 cells were seeded into separate 96-well plates and treated with different doses of VIT for 24, 48, and 72 h, respectively. Then, the cells were withdrawn from the media and incubated with Cell Counting Kit-8 (CCK-8) (CT0001, Sparkjade, Qingdao, China) reagents at 37 °C for 4 h. Thereafter, the absorbance of each well was instantly measured at 450 nm using an ELISA microplate reader (TECAN Infinite F50, Männedorf, Switzerland). The cell viability rates were calculated after normalizing them to the control group.

MDA-MB-231 and HCC-1937 cells were incubated with VIT for 24 hours, while groups without VIT were established as the corresponding negative controls. Secondly, cells were rinsed twice with pre-chilled PBS, and then stained with the Annexin V Apoptosis Detection kit (640932, BioLegend, San Diego, CA, USA). Finally, the cells were analyzed by flow cytometry. Apoptosis was determined based on the percentage of cells binding with Annexin V+/PI+. The results are the average values obtained from three separate experiments.

After treatment with VIT for 24 h, MDA-MB-231 and HCC-1937 cells were collected and washed with pre-chilled PBS before being fixed overnight at 4 °C with 70% ice-cold ethanol. After washing with PBS, the cells were stained with a solution containing 50 µg/mL PI and 1 mg/mL RNase A and incubated in the dark for 30 min at 37 °C. Analysis of the cell cycle distribution was performed by flow cytometry.

To perform the pull-down assay, VIT (5 µmol) was coupled to CNBr-activated Sepharose 4B (GE Healthcare Bio-Sciences Corp,, Piscataway, USA) matrix beads (0.1 g) in 500 µL 0.1 M NaHCO${}_3$ pH 8.3 solution containing 0.5 M NaCl and 40% DMSO (pH 8.0) and incubated overnight at 4 °C, in accordance with the manufacturer’s instructions. Then, the total MDA-MB-231 cell lysates were incubated overnight in the reaction buffer (50 mM Tris pH7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP-40, 2 µg/mL BSA) with either Sepharose 4B–VIT or Sepharose 4B alone, with gentle rocking at 4 °C. Finally, the binding was determined by Western immunoblotting.

MDA-MB-231 and HCC-1937 cells were treated with VIT for 24 h. The following day, the cells were harvested, washed with ice-cold PBS, and lysed on ice for 15 min in RIPA lysis buffer containing protease inhibitors. Then, the lysates were centrifuged at 12,000 g for 20 min at 4 °C. The protein concentrations were measured using a BCA assay kit (P0010, Beyotime, Shanghai, China). Equal amounts of total protein were loaded and separated on an 8% SDS-PAGE gel before being transferred to a PVDF membrane. The blots were blocked in 5% non-fat milk for 1 hour at room temperature and then incubated with primary antibodies targeting either CREBBP, HSP90AA1, or HIF-1A, overnight at 4 °C. Subsequently, the blots were incubated with HRP (horseradish peroxidase)-conjugated secondary antibodies. Finally, the protein bands were visualized using an electrochemiluminescence detection system (Odyssey® Fc, Lincoln, Nebraska, USA). The levels of the detected proteins were normalized to $\beta$-actin.

Statistical analysis was conducted using Origin 8.0 (Northampton, Massachusetts, USA), and the data are presented as mean $\pm$ SD. The t-test was used for pairwise comparison, with statistical significance considered as p 0.05 (*) and as highly significant at p 0.01 (**).

We conducted an analysis to identify potential VIT target genes for the treatment of TNBC. Firstly, to investigate the potential targets of VIT in TNBC, we utilized the Super-PRED database, which predicted 105 candidate genes (Supplementary File 1) as potential targets for VIT. In addition, we retrieved a list of 1588 TNBC-related targets from the DisGeNET database (Supplementary File 2).

A total of 37 overlapping targets were identified by expressing these two target clusters in a Venn diagram (Fig. 1B and Table 1). To visualize the relationship between VIT and the TNBC-related targets, we constructed a network known as the ‘VIT–TNBC-related targets’ network (Fig. 1C), which consisted of 38 nodes that represented the genes and 37 edges that depicted the connections between these genes.

To gain further insights into the functions of the 37 potential VIT target proteins, we performed GO enrichment and KEGG pathway analyses. The GO enrichment analysis identified a link between the proteins and important biological processes, such as transcription regulation, protein phosphorylation, neutrophil degranulation, peptidyl-threonine phosphorylation, and extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) cascade regulation. In terms of cellular components, these proteins were found to be located in various areas, including the cytoplasm, cell surface, ficolin-1-rich granule lumen, nucleoplasm, and extracellular exosome. The proteins were primarily involved in functions such as transcription factor binding, protein serine/threonine kinase activity, Adenosine triphosphate (ATP) binding, protein kinase binding, and protein kinase activity, among others in the molecular function category (Fig. 1D).

The KEGG pathway analysis revealed that VIT in TNBC was associated with 67 signaling pathways (p 0.05), with several of the top 20 pathways (Fig. 1E) found to be closely linked to tumor biology, including the HIF-1 signaling pathway, cell cycle regulation, autophagy, PD-L1 expression and the PD-1 checkpoint pathway in cancer, along with the thyroid hormone signaling pathway.

To illustrate the VIT hub targets and the effects on TNBC, we constructed a PPI network using the STRING database to obtain the potential protein interactions. As shown in Fig. 1F, HIF-1A, CREEBP, and HSP90AA1 were selected as the VIT hub genes by analyzing the degrees between nodes.

To verify the reliability of the interaction between VIT and its potential hub genes, Libdock software was used to perform molecular docking. As depicted in Fig. 2A, we found that VIT forms hydrogen bonds with specific amino acid residues (ASN51, LYS58, ASP93, GLY97, and ASP102) in HSP90AA1, while also exhibiting attractive charge interactions with LYS58 in HSP90AA1. One carbon atom in VIT expressed the potential to form C–H bonds with the MET1133 amino acid residue in CREBBP (Fig. 2B). Analysis of the binding sites between VIT and HIF-1A demonstrated that VIT bound to the hydrophobic cavity of HIF-1A, with presented hydrogen bond featuring particularly prominently among the many interactions (PHE100, THR196, and GLN203) (Fig. 2C). Therefore, these results suggest that VIT might mainly act on HIF-1A, HSP90AA1, and CREEBP hub targets in treating against TNBC.

Fig. 2.

Molecular docking validation and pull-down assay on potential VIT target genes in TNBC. (A) Intermolecular interactions by VIT with active site residues in HSP90AA1. (B) Intermolecular interactions by VIT with CREBBP active site residues. (C) Intermolecular interactions by VIT with HIF-1A active site residues. (D–F) The MDA-MB-231 cell lysates were incubated with Sepharose 4B-VIT beads and the pull-down proteins were analyzed by Western blot.

To further identify the potential direct targets of VIT, Sepharose 4B–VIT beads were used in pull-down assays with MDA-MB-231 cell lysates. These results showed that VIT possessed a direct affinity to HSP90AA1 and HIF-1A but not with CREBBP (Fig. 2D–F).

To explore the clinical value of these three VIT hub target genes in TNBC, the Kaplan–Meier plotter online database was used to analyze disease-free survival. As shown in Fig. 3, the high expression of HSP90AA1, CREBBP, and HIF-1A was associated with poor overall survival (OS) for TNBC patients. These results found that the expression levels of HSP90AA1, CREBBP, and HIF-1A were significantly associated with the clinical prognosis of TNBC and may play key roles in the pathogenesis of TNBC.

Fig. 3.

The overall survival (OS) analysis of HSP90AA1, HIF-1A, and CREBBP by Kaplan–Meier. (A) The OS analysis for HIF-1A. (B) The OS analysis for HSP90AA1. (C) The OS analysis for CREBBP.

To evaluate the effect of VIT on cell proliferation, we performed a CCK8 assay using TNBC cell lines MDA-MB-231 and HCC-1937, which were treated with varying concentrations of VIT (ranging from 0 to 80 µM) for 24 h, 48 h, and 72 h. The results showed that the cell viabilities of the MDA-MB-231 and HCC-1937 cell lines were decreased by VIT treatment in both a dose- and time-dependent manner (Fig. 4A,B, Supplementary Fig. 1). The IC${}_{50}$ values for VIT in the in MDA-MB-231 cells were determined at 24 h, 48 h, 72 h as 21.57 µM, 4.8 µM, and 4.8 µM, respectively. Whereas the respective values in the HCC-1937 cells were 31.60 µM, 3.86 µM, and 3.5 µM. Moreover, VIT presented with no cytotoxicity in normal HEK293T cells (Supplementary Fig. 2). Thus, these results indicate that VIT effectively inhibits the proliferation of TNBC cells in a concentration-dependent manner. In addition, the pharmacokinetic results found that many flavonoids reached maximum absorption at 24–36 h [10]. Therefore, we chose 1.25 µM, 5 µM, and 20 µM VIT concentrations to treat the MDA-MB-231 cells and concentrations of 3 µM, 10 µM, and 30 µM to treat the HCC-1937 cells, with a treatment period of 24 h chosen for both cell lines for future experiments.

Fig. 4.

Cell viability in MDA-MB-231 and HCC-1937 cells treated with VIT. (A) The effect of VIT on MDA-MB-231 cell growth. (B) The effect of VIT on HCC-1937 cell growth. The viability for each cell line was calculated using the Cell Counting Kit-8 (CCK-8) assay.

The ability of VIT to induce apoptosis in MDA-MB-231 and HCC-1973 cells was detected by an Annexin V/ PI apoptosis assay using flow cytometry. In Fig. 5A–D, VIT efficiently induced apoptosis in both MDA-MB-231 and HCC-1937 cell lines in a dose-dependent manner. Apoptosis was observed in 20 µM (22.81%) VIT-treated MDA-MB-231 cells and in 30 µM (18.35%) VIT-treated HCC-1937 cells.

Fig. 5.

Apoptosis and cell cycle analyses in MDA-MB-231 and HCC-1937 cells treated with VIT by flow cytometry. (A,B) The effect of VIT on apoptosis in MDA-MB-231 cells. (C,D) The effect of VIT on apoptosis in HCC-1937 cells. (E,F) The effect of VIT on cell cycle progression in MDA-MB-231 cells. (G,H) The effect of VIT on cell cycle progression in HCC-1937 cells.

Then, we used PI staining to investigate whether VIT affects cell cycle progression. Fig. 5E,F demonstrate that the G2-phase distribution for MDA-MB-231 cells increased significantly as the VIT concentration increased and was accompanied by a decrease in the G1-phase distribution. This same situation was also reflected by HCC-1937 cells. In Fig. 5G,H, it can be seen that the G2-phase distribution for HCC-1937 cells also markedly increased, while the increase was more obvious in the initial low-concentration group. Moreover, the G2-phase distribution increased continuously as the VIT concentrations increased, while the G1-phase distribution significantly decreased. However, 30 µM of VIT simultaneously increased cell cycle arrest in the G2 phase and S phase. The results showed that VIT induced cell cycle arrest in the G2 phase in both the MDA-MB-231 and HCC-1937 cell lines.

Based on the results of the network pharmacology and molecular docking, we investigated whether VIT inhibited cell proliferation in TNBC through the hub genes; thus, we performed Western blotting to measure the expression of HSP90AA1, CREBBP, and HIF-1A in the MDA-MB-231 and HCC-1937 cell lines following incubation with VIT. As shown in Fig. 6, the protein levels of all three targets were significantly downregulated in the MDA-MB-231 and HCC-1937 cells after VIT treatment, in a concentration-dependent manner. These results illustrated that VIT inhibits the growth of TNCB cells by regulating its potential targets HSP90AA1, CREBBP, and HIF-1A.

Fig. 6.

Protein expression of CREBBP, HSP90AA1, and HIF-1A in VIT-treated cells. (A) MDA-MB-231 cells were treated with 1.25, 5, and 20 µM VIT for 24 hours to assess the expressions of CREBBP, HSP90AA1, and HIF-1A by Western blot analysis. (B–D) Relative expressions of CREBBP, HSP90AA1, and HIF-1A were analyzed by software Image J 1.54d (Wayne Rasband and contributors National Institutes of Health, USA). ** p 0.01, * p 0.05 vs. untreated group. (E) HCC-1937 cells were treated with 3, 10, and 30 µM VIT for 24 hours to assess the expressions of CREBBP, HSP90AA1, and HIF-1A by Western blot analysis. (F–H) Relative expressions of CREBBP, HSP90AA1, and HIF-1A were analyzed by Image J. ** p 0.01, *p 0.05 vs. untreated group.