Drug preparation
Codonopsis pilosula Nannf., Poria cocos Wolf., Epimedium brevicornu Maxim., Prunella vulgaris L, Curcuma phaeocaulis Val., and Scutellaria barbata D. Don. were provided by Shanghai Kang Qiao Chinese Cut Crude Drug Company and identified by WUXI APPTEC (SHANGHAI) Co., Ltd (China). Morphological, microscopic, and phytochemical identification were performed in accordance with the Pharmacopoeia of the People’s Republic of China (2015 edition). Codonopsis pilosula Nannf. (12 g), Poria cocos Wolf. (12 g), Epimedium brevicornu Maxim. (15 g), Prunella vulgaris L. (9 g), Curcuma phaeocaulis Val. (30 g), and Scutellaria barbata D. Don. (30 g) of XLLXF were weighed and added to a stainless pot. After 1080 mL of distilled water was added and boiled for 1 h, liquid was collected. Then, 1080 mL of water was added and boiled for another 1 h to collect liquid again. The solution was combined twice, a rotary evaporator was used to concentrate the solution, and the final volume was concentrated to 500 ml. The solution was combined and concentrated twice, and the concentrated extract was freeze dried to obtain the XLLXF extract at a yield of 29.23% (w/w, dried extract/crude herbs).
Collection of active ingredients and targets of XLLXF
The active ingredients of Codonopsis pilosula Nannf., Poria cocos Wolf., Epimedium brevicornu Maxim., Prunella vulgaris L, Curcuma phaeocaulis Val., and Scutellaria barbata D. Don. in XLLXF were searched through the TCMSP database (https://tcmspw.com/tcmsp.php). Curcuma phaeocaulis Val. was used, with oral bioavailability (OB) ≥ 30% and drug likeness (DL) ≥ 0.10 as the screening conditions, while the others had screening conditions of OB ≥ 30% and DL ≥ 0.18. Relevant databases, such as CNKI and PubMed, were consulted to supplement the key herbal-related active ingredients screened in the TCMSP database. TCMSP was also used to obtain related targets, and then gene symbols were acquired from UniProt database (https://www.uniprot.org).
Collection of targets between XLLXF and TNBC
With “triple negative breast cancer” as the search term, the TNBC-related targets were selected by the Gene Cards database (https://www.genecards.org). A Venn diagram was drawn to clarify the interaction between the TNBC-related targets and the potential targets of XLLXF.
Construction of “XLLXF–active ingredients–targets” network and PPI network
The “XLLXF–active ingredients–targets” network was drawn by Cytoscape 7.2. Its built-in tool network analyzer was used to calculate the degree, and the main active ingredients were screened out. The PPI network was constructed using the string database (https://string-db.org). Modular clustering of the protein network was conducted to obtain core proteins with higher degrees by using the MCODE plug-ins in Cytoscape 7.2.
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
GO and KEGG pathway enrichment analysis was performed through the DAVID database (https://david.ncifcrf.gov). On the basis of gene counts, the top 10 processes were correspondingly shown as bubble charts. The “XLLXF–active ingredients–targets–key signal pathway” network was drawn by Cytoscape 7.2.
Molecular docking
The 3D crystal structures of the core targets were extracted from the Protein Data Bank (http://www.rcsb.org/pdb/). Then, the protein structures were processed by AutoDock Tools, including removal of ligands and water molecules, calculation of Gasteiger charge, addition of polar hydrogen, and combination of non-polar hydrogen. Subsequently, molecular docking was carried out via AutoDock Vina. Finally, the receptor–ligand complex was imported into Ligplus software to analyze the hydrogen bonding and hydrophobic interaction between the receptor and the ligand.
Cell lines and cell proliferation assays
Human TNBC cell line MDA-MB-231 was purchased from the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle medium (DMEM) containing with 10% fetal bovine serum and 1% penicillin/streptomycin in a saturated humidity environment at 37 °C and 5% CO2
The proliferation of MDA-MB-231 cells was detected by 3-(4,5)- dimethylthiazolyl-3,5-diphenyltetrazolium bromide (MTT) assay. In brief, the cells were blown into a single-cell suspension (1.0 × 105 cells/mL) and seeded into 96-well plates (100 µL/well). Twenty-four h later, the culture medium was replaced with fresh medium containing various concentrations of XLLXF (0, 6.25, 12.5, 25, 50, 100, and 200 µg/mL). The cells were then cultured with a drug-containing medium for 24, 48, and 72 h. Afterwards, the MTT solution was added into the 96-well plates and incubated for 4 h at 37 °C. Optical density (OD) was detected at 490 nm with a BioTek instrument (Winooski, Vermont, USA). Data were expressed as the mean ± SD of at least three independent experiments.
Colony formation assays
Colony formation assays were performed to further determine the inhibitory effect of XLLXF on the tumorigenicity of TNBC cells. A total of 500 MDA-MB-231 cells were seeded into six-well plates to incubate overnight. The cells were incubated with different final concentrations (0, 50, 100, and 200 µg/mL) of XLLXF for 7 days. After fixing with 4% paraformaldehyde and staining with a crystal violet solution, colonies containing more than 30 individual cells were counted under a stereomicroscope.
Cell apoptosis assay
The apoptosis in MDA-MB-231 cells was measured by TUNEL and JC-1 with the use of a one-step TUNEL apoptosis assay kit (Beyotime, China) and a mitochondrial membrane potential assay kit with JC-1 (Beyotime, China). Log-phase MDA-MB-231 cells were seeded in a six-well plate with small glass slides in advance and randomly divided into control group and XLLXF groups (50, 100, and 200 µg/mL). After treatment for 24 h, TUNEL staining was performed in the TUNEL experiment. One mL of JC-1 staining working solution was added in the JC-1 experiment and incubated at 37 °C for 30 min in a cell incubator. Then, the cells were washed twice with JC-1 staining buffer and added with 2 mL of DMEM. Images were captured by a confocal laser microscope.
Phalloidin dying of F-actin
Log-phase MDA-MB-231 cells were harvested and resuspended. A total of 200 µL of suspended cells was then pipetted into 35 mm chamber slides at a density of 1 × 105/mL. After being subjected to treatments with DMEM or XLLXF (100 µg/mL) for 24 h, the cells were fixed with 4% paraformaldehyde in cytoskeleton for 30 min at room temperature. Then, they were permeabilized with 0.5% Triton X-100 for 5 min and blocked with BSA for 1 h at room temperature. Between each step described above, the cells were washed three times with PBS at 5 min each. F-actin was stained with Alexa Fluor 488 Phalloidin for 1 h to visualize the actin cytoskeleton. The cells were then counterstained with 4 mg/mL of 4,6-diamidino-2-phenylindole (DAPI).
Immunofluorescence experiment
The treated MDA-MB-231 cells were fixed in 4% paraformaldehyde for 5 min, permeabilized with 0.5% Triton X-100 for 5 min, and blocked with BSA for 1 h at room temperature. Then, they were incubated with primary antibodies at 4 °C overnight, followed by incubation with fluorophore-conjugated secondary antibody for 1 h. The samples were stained with DAPI and imaged using a confocal microscope after washing three times.
Three-dimensional cultures
A 24-well tissue culture plate was evenly coated with 200 µL/well growth factor-reduced Matrigel, which was allowed to solidify at 37 ℃ for 60 min before cells were plated. The cell suspension was added (1 × 105 cells/well) onto the surface of the Matrigel and incubated at 37 ℃ for 24 h. Net-like structures lined with tumor cells were considered the mimicking vessels.
Western blot analysis
After the MDA-MB-231 cells were treated with XLLXF (50, 100, and 200 µg/mL) for 24 and 48 h, total cell protein lysates were extracted using RIPA lysis buffer that contained protease and phosphatase inhibitor cocktails. Protein lysates (20 µg), which were determined by BCA analysis (Beyotime, China), were loaded onto 10% SDS-PAGE gels. The protein bands were transferred onto NC membranes and blocked with 5% non-fat milk for 1 h at room temperature. The NC membranes with proteins were incubated with diluted primary antibodies at 4 °C overnight. The primary antibodies used in the analyses were as follows: VEGFA (1:1,000, Proteintech), MMP2 (1:1000, Proteintech), MMP9 (1:1000, Cell Signaling Technology), Vimentin (1:1000, Proteintech), VE-cadherin (1:1,000, Cell Signaling Technology), TIMP-1 (1:1000, Proteintech), TIMP-3 (1:1,000, Proteintech), and Twist1 (1:1000, Proteintech). The membranes were incubated with relative sources of secondary antibodies (1:5000) at room temperature for 1 h. Specific protein bands were recognized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA). Image J software was used for image analysis.
Tumor xenograft assay in vivo
Seven-week-old female nude BALB/c mice (18–20 g) were obtained from Shanghai SLAC Laboratory Animal Technology Co., Ltd (Shanghai, China). The protocol was approved by the Animal Research Ethics Committee of Shanghai University of Traditional Chinese Medicine. MDA-MD-231 cells (1 × 107) premixed with Matrigel at a ratio of 1:1 was subcutaneously injected into the fourth pair of breast fat pads on the left side of each mouse. The tumors formed approximately 7 days after the inoculation. XLLXF (18 g/kg) was administered intragastrically; it was converted in accordance with the 10-fold effective dose between human (60 kg) and mice (20 g). All mice were then randomly divided into two groups (n = 5): control group (i.g., saline) and XLLXF group (i.g., 18 g/kg). The mice received corresponding treatment once a day for 4 weeks. Tumor volumes were calculated using the following formula: V = 0.5 × a × b2, where V denotes tumor volume, a denotes maximum tumor diameter, and b denotes minimum tumor diameter. Body weight was measured once a week as an indicator to assess the animals’ overall health. The mice were euthanized upon the experiments’ termination.
Electron microscopy
Xenograft tissues (0.5 mm3) were fixed in cold 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer and postfixed in 1% osmium tetroxide, dehydrated, and embedded in a standard fashion for transmission electron microscopy. The specimens were then embedded, sectioned, stained, and observed using Hitachi HT-7700 TEM.
Immunohistochemical (IHC) staining
The slides were deparaffinized twice with xylene for 10 min and rehydrated with 100–75% ethanol for 10 min. After the slices were washed with PBS three times, they were boiled in 10 mm sodium citrate buffer solution for 8 min for antigen repair. Sections were permeabilized with 3% hydrogen peroxide dissolved in methanol at room temperature in the dark to eliminate endogenous peroxidase activity and then blocked by 10% goat serum to reduce nonspecific binding. The samples were then washed with PBS three times and incubated with 1:200 diluted primary antibodies in a humid chamber at 4 °C overnight, followed by incubation with a 1:200 dilution of biotinylated secondary antibodies. Immediately thereafter, 3,3-diaminobenzidine substrate was applied for color development, and counterstaining with Mayer’s hematoxylin was performed.
PAS-CD34 dual staining
The procedure was the same as the above description in the IHC part. In brief, after DAB reaction, sections were treated with 0.5% periodic acid solution for 10 min and rinsed with distilled water for 5 min, followed by staining in Schiff solution for 15–30 min. After the sections were rinsed with distilled water, they were counterstained with hematoxylin, dehydrated, cleared, and mounted.
ELISA
The levels of VEGFA and MMP2 were determined by ELISA kits (mlbio, shanghai, China). The serum of mice was specifically collected; 20 µL of serum, 30 µL of sample dilution, and 100 µL of HRP were added to each well of the antibody-coated plate for 1 h at 37 °C. Then, the liquid was removed, and the microplate was washed three times. Afterwards, 50 µL of the reaction solution was added to each well for 30 min at 37 °C with light prevention. Next, 50 µL of stop solution was added, and the absorbance was measured at 450 nm.
Statistical analyses
SPSS software (version 25.0) was used for statistical analysis, and GraphPad Prism software (version 8) was used for plotting. All images were measured by ImageJ software. Measurement data conformed to a normal distribution, and they were compared via independent sample t-test analysis for two groups. One-way ANOVA was used for multigroup analysis. Nonparametric rank sum test was used if the measurement data did not conform to a normal distribution. P < 0.05 was considered statistically significant.