Herbal extraction
XJP consisted of She Xiang (Moschus), Mu Bie Zi (Cochinchina Momordica), Cao Wu (Kusnezoff Monkshood Root), Feng Xiang Zhi (Resina Liquidambaris), Ru Xiang (Olibanum), Moyo (Myrrh), Wu Ling Zhi (Trogopterus Dung), Dang Gui (Chinese Angelica), Di Long (Phertima), and Xiang Mo (Pine-soot ink). All the herbals were purchased in the Affiliated Hospital of Shandong University of Traditional Chinese Medicine, and all of them were identified by Chuanjiang Ma, who was devoted to the research of traditional medicine. The ratio of these 10 herbs was 1:5:5:5:2.5:2.5:5:2.5:5:0.4. The extract of XJP was prepared by decocting the dried prescription of herbs. Briefly, raw materials of XJP formulation were mixed and crushed into small pieces. Ten-time volumes of water were added with raw components and boiled for 2 h. Then, a centrifuge (Eppendorf, Germany) was used to precipitate at 3000 rpm for 5 min, five times. The decoction was concentrated by removing a portion of water in a vacuum at 60℃. Finally, the concentration of XJP was 1 g/mL. Before analysis, the decoction was filtered through a 0.22-µm polytetrafluoroethylene filter (Millipore, MA, USA). The filtered decoction was stored at 4℃ before treating cells.
Cell culture
Human breast cancer cell lines MDA-MB-468 (kindly provided by Professor Changgang Sun, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, Shandong) and MDA-MB-231 (kindly provided by Professor Fukai Wang, Affiliated Cancer Hospital of Shandong First Medical University, Jinan, Shandong) were cultured in Dulbecco’s modified Eagle’s medium (cat. no. CM15019; Macgene, China) with 10% fetal bovine serum (FBS, cat. no. 04-001-1ACS; Biological Industries, Israel), penicillin (100 U/mL), and streptomycin (100 mg/mL) (cat. no. cc004; Macgene, China). The cells were incubated at 37°C with 5% CO2 in a humidified atmosphere. All experiments were performed on logarithmically growing cells.
Reagents
Epirubicin (cat. no. E122334; Aladdin, China) was dissolved in dimethyl sulfoxide (cat. no. D8371; Solarbio; China). CCK-8 (cat. no. CK04) was purchased from Dojindo Molecular Technologies, Inc.. Antibodies against E-cadherin (CDH1; cat. no. 22018-1-AP), β-actin (ACTB; cat. no. 66009-1-Ig), β-catenin (CTNNB1; cat. no. 51067-2-AP), N-cadherin (CDH2; cat. no. 22018-1-AP), and caspase-3 (CASP3; cat. no 66470-2-Ig.) were purchased from Proteintech Group, Inc. Antibodies against MMP2 (cat. no. ab86607), MMP9 (cat. no. ab137867), and caspase-9 (CASP9; cat. no. ab202068) were obtained from Abcam, Inc. Caspase-8 (CASP8; cat. no.4790) and Parp (PARP1; cat. no.9532) were purchased from Cell Signaling Technology. Secondary antibodies horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (H + L) (cat. no. SA00013-4) and HRP-labeled goat anti-mouse IgG (H + L) (cat. no. SA00001c1) were purchased from Proteintech Group, Inc.
Cell viability assay
The sensitivity of breast cancer cells to XJP and epirubicin was detected using the Cell Counting Kit-8 (CCK-8) following the manufacturer's protocols. Briefly, MDA-MB-231 (6.0 × 103 cells/well) and MDA-MB-468 (1.0 × 104cells/well) cells were cultured in quintuplicate in 96-well plates at 37°C overnight and treated with the drugs at different concentrations: [epirubicin, MDA-MB-231 (0, 2.5, 5, 10, 20, and 40 μg/mL) and MDA-MB-468 (0, 0.0625, 0.25, 1, 4, and 16 μg/mL)]; [XJP, MDA-MB-231 (0, 2.5, 5, 10, 20, and 40 mg/mL), and MDA-MB-468 (0, 5, 10, 20, 40, and 80 mg/mL)] for 48 h. Subsequently, the medium was removed, and the cells were incubated with 90 μL of new culture medium supplemented with 10 μL of CCK-8 reagents for 40 min. The absorption at 450 nm was detected using a spectrophotometer (Type, 1510; Thermo Fisher Scientific Inc.).
Flow cytometric analysis
Cell apoptosis was assessed using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide kit following the supplier’s protocols. The cells were seeded at a density of 3 ´ 105 cells/well in six-well plates. After 24 h of culture, different concentrations of XJP [MDA-MB-231 (0, 17, and 34 mg/mL) and MDA-MB-468 (0, 5, and 10 mg/mL)] were added to the plates, and the cells were further cultured for 48 h. After incubation, the cells were collected. Then, 5 µL of FITC and 5 µL of Polyimide (PI) were added per well. The cells were then analyzed using the Fluorescence Activating Cell Sorter (FACS) Calibur system (BD Company, NJ, USA) in 15 min.
Cell migration and invasion assay
Twenty-four-well plates were used to determine the effect of drugs on the migration and invasion of breast cancer cells. Breast cancer cells were harvested and resuspended in the serum‑free medium supplemented with drugs at different concentrations (XJP for MDA-MB-231 cells at 0, 3.75, and 7.5 mg/mL; and for MDA-MB-468 at 0, 2, and 4 mg/mL). An 8-μm-pore-size filter (cat. no. 353097; BD Biosciences,NJ, USA) was pre-coated with Matrigel (cat. no. 354234; BD Biosciences) at 37°C for 30 min (Matrigel was abandoned for migration assay), and the lower chamber was filled with 500 µL of the medium containing 10% FBS. Suspended breast cancer cells (5 ´ 104 cells/well in 400 µL of medium) were seeded in the upper chamber and incubated for 48 h at 37°C. Subsequently, the filter was fixed with 4% paraformaldehyde solution (cat. no. P0099; Beyotime Institute of Biotechnology) for 20 min and stained with 0.1% crystal violet for 30 min at room temperature. Finally, the cells and Matrigel on the upper chamber were scraped with a cotton swab, and the invasive cells on the lower surface were counted under an inverted fluorescence microscope (Vert. A1; Carl Zeiss AG). Three fields of each sample were captured at 100× magnification.
Western blot analysis
The total proteins were obtained from breast cancer cells treated with XJP for 48 h at different concentrations [MDA-MB-231 (0, 3.75, and 7.5 mg/mL for invasion and migration, and 0, 17, and 34 mg/mL for apoptosis); MDA-MB-468 (0, 2, and 4 mg/mL for invasion and migration, and 0, 5, and 10 mg/mL for apoptosis)] The cell lysates were acquired using radio-immunoprecipitation assay (RIPA) cell lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) and centrifuged at 13,200 rpm at 4°C for 15 min. A bicinchoninic acid assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology) was used to determine the protein concentration, and 10% sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) was used to separate equal amounts of proteins (30 μg protein loaded per lane). After the proteins were transferred to Polyvinylidene fluoride (PVDF) membranes (cat. no. Ipvh00010-01; Millipore, MA, USA), the membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated with primary antibodies at a 1:1000 dilution at 4°C overnight. Appropriate secondary antibodies conjugated with horseradish peroxidase at 1:5000 dilution were used to incubate membrane-bound primary antibodies at room temperature for 1 h, followed by the development of immunoblots using an enhanced chemiluminescence kit (cat. no. WBKLS0050; Merck KGaA). The densitometry analysis was performed using the ImageJ version. 1. 52a (Media Cybernetics Inc.).
Immunofluorescence
Breast cancer cells were plated in 24-well plates containing coverslips. When the cells reached 60%-70% confluence, the media were removed and the cells were fixed with 4% paraformaldehyde for 15 min. Permeabilized cells were treated with 0.1% Triton X-100 (Solarbio, China) for 10 min, followed by blocking with 3% bovine serum albumin (Hat Biotech, China) for 1 h. Then, the cells were incubated with a primary antibody against Recombinant Human Catenin beta-1 (CTNNB1) in blocking buffer at 4℃ overnight. Next, the cells were incubated with fluorescein isothiocyanate (FITC)- or tetramethylrhodamineisothiocyanate (TRITC)-conjugated secondary antibody and 4',6-diamidino-2-phenylindole (DAPI, Solarbio, China) at room temperature for 2 h. The coverslips were sealed with the slides using fluorescence decay-resistant medium (Hat Biotech, China).
Statistical analysis
All the experiments were carried out in triplicate (n = 3) unless otherwise mentioned, and the data were presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0.1 (244) software (GraphPad Software, Inc.) by one-way analysis of variance with Dunnett's post hoc test. A P value <0.05 indicated a statistically significant difference.