2.1. Cell culture
MDA-MB-231, MCF-7 and human umbilical vein endothelial cells (HUVECs) were purchased from the Wuhan Punuosai Life Technology Co., Ltd. (Wuhan, China) and Shanghai Suran Biological Technology Co., Ltd. (Shanghai, China). All cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM), and high glucose was supplemented with 10% fetal bovine serum (FBS, Gibco) in a humidified atmosphere of 5% CO2 at 37˚C. Eupafolin (purity ≥ 99%) was purchased from Yuanye Biotechnology. In this study, Eupafolin was dissolved in dimethyl sulfoxide (DMSO, Beijing Solarbio Science & Technology Co., Ltd) at different concentrations (0, 25, 50 and 100 µM). Subsequently, cells were treated with Eupafolin for different time periods. The siRNA-Cav-1(Suzhou Jima) was transfected into breast cancer cells using Lipofectamine 2000™ reagent (Suzhou Jima). Rapamycin (RA) and 3-methyladenine (3-MA) were purchased from (MCE).
2.2. Cell counting kit-8 and colony formation assay
In brief, 5,000 cells were seeded onto 96-well plates, treated with 0, 25, 50 and 100 µM Eupafolin, and then incubated for 24, 48 and 72 h. Then, a microplate reader was utilized to measure the absorbance at 450 nm (TECAN). Then, cells with an adhesion rate > 90% (in logarithmic growth phase) were trypsinized, centrifuged, and resuspended for counting cells. Then, the cells were placed into 6-well plates at a density of 1,000 cells/well, and then placed into a 37˚C CO2 incubator for culturing. After 7 days of incubation, once the colonies were visible to the naked eye, they were removed from the 6-well plate, the culture medium was discarded, and 2 mL of methanol was added to each well for 30 minutes. Next, the methanol was discarded, and 1 ml of 0.1% crystal violet was added to each well for staining for three minutes. Finally, the purple crystals were washed away, and photos were taken with a digital camera. The colonies were counted under a light microscope.
2.3. Transwell experiments.
After Eupafolin treatment, cells were digested, resuspended in a serum-free medium, and adjusted to a density of 1x105 cells/ml. Next, the transwells were placed in an incubator for 24 h. After that, the cells were removed and the medium was aspirated. In a new 24-well plate, 600 µl of 4% paraformaldehyde was added. The transwell was placed at 37˚C and cells were fixed for 30 min. The fixative solution was then removed and cells were stained at 37˚C using 0.2% crystal violet for 10 min. Then, the excess crystal violet was removed prior to microscopy. After the cells were dried, They were observed and counted under an inverted light microscope using a digital camera (magnification, x80). The cell invasion test procedure was then used similarly to a cell migration assay.
2.4. Cell cycle and apoptosis analysis
Apoptosis was quantified using the Annexin-V/ FITC apoptosis detection kit, according to the manufacturer’s instructions (Ebisson). After drug treatment for 24 h, breast cancer cells and HUVECs were typsinized and then centrifuged at 800 x g for five min. Then, the supernatant was discarded, resuspended in 300 µl 1X binding buffer, and a 5 µl sample was mixed with FITC-Annexin V. Then, 5 µl PI staining solution was added onto 200 µl 1X binding buffer five min prior to detection. Flow cytometry was conducted using FACS (Thermo Fisher Scientific, Inc). For cell cycle analysis, cells were collected and fixed in 70% ethanol at 4˚C overnight. Next, cells were resuspended in 500 µl 1X PI solution (Baihao) for 30 min at 37˚C. The analysis was then performed through the use of FACS (Thermo Fisher Scientific, Inc.). Finally, the results were assessed using ModFit LT (version 5.0; Verity Software House, Inc.).
2.5. Reverse transcription-quantitative PCR (RT-qPCR).
According to instructions provided with the kit, the TRIzol reagent (Ruan) was utilized to extract total cellular RNA, and cDNA was generated through the use of Fastking RT kit (Tian gen Biotech Co., Ltd) with 2 µg of RNA. The primers used were designed using National Center for Biotechnology Information (NCBI). A RT-qPCR detection system (Eppendorf) was used to perform the RT-qPCR reactions using SYBR Green (Tian gen Biotech Co., Ltd.) and a total of 20 µl of reaction mixture. The 2−ΔΔCq method  was utilized to assess gene expression.
2.6. Angiogenesis analysis
The HUVECs were seeded onto a six-well plate. After 12 hours, they were treated with Eupafolin at concentrations of 50 µM and 100 µM for 24 hours. Then, we added matrigel to the 96-well plate (40 ml/well) and maintained it for 60 minutes. The cells were then digested with trypsin and diluted to 2×105 cells/ml. Then, 100 µl of the cell suspension was added into each well of a 96-well plate. The cells were incubated at 37˚C for 6h, and then visualized using randomly select several fields of view under an inverted microscope to observe the microtubule structure.
2.7. Western blotting
After the cells were treated with Eupafolin for 24 h, they were harvested, and lysed on ice with RIPA lysis buffer for 30 min. Next, the BCA protein assay kit was utilized to determine the protein concentration. Furthermore, 5X loading buffer was added and the proteins were denatured at 95˚C for 10 min. Then, the protein sample was added to each well, separated using 10–12% SDS-PAGE at 120 V, and transferred to PVDF membranes. The membraned were probed with primary antibodies against Bcl-2 (cat. no. 4223), Bax (cat. no. 2772), cleaved caspase-3 (cat. no. 9661), PI3K (cat. no. 4257), p-PI3K (cat. no. 17366), Akt (cat. no. 4691), p-Akt (cat. no. 4060), LC3B (cat. no. 43566), Beclin-1 (cat. no. 3495), Caveolin-1 (cat. no.3267), CDK2 (cat. no.2546), CDK4 (cat. no.12790), Cyclin B1 (cat. no.4138) and GAPDH (cat. no. 5174) (all 1:1,000, Cell Signaling Technology, Inc.) at 4˚C overnight. Subsequently, the membranes were washed three times with PBS, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1h at 37˚C. The protein bands were visualized using chemiluminescence assay kit (Dalian Meilun Biotechnology Co., Ltd.) and visualized with an imaging system (Tanon).
2.8. RNA Interference
Cav-1 siRNAs oligonucleotides were synthesized by Suzhou Gene Pharma Co., Ltd. (Suzhou, China). After breast cancer cells were seeded for 12h, Cav-1 siRNA and lipo2000 were diluted with serum-free medium and added to the cells. The medium was changed to complete medium after 6h, and the cells continued to culture in the 37˚C CO2 incubator.
2.9. Experimental Animal
All the experiments were approved by Animal Care and Use Committee of Jilin University (Grant No. SY202012006) and were carried out in compliance with the ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357). Overall, 10 female BALB/c nude mice (5 weeks old) were purchased from the Liaoning Changsheng Laboratory Animal Technology Co, Ltd. Then, 5×106 MCF-7 cells in 100 ml PBS were subcutaneously inoculated into armpit of nude mice. Seven days after injection of cells, when the tumor had grown to approximately 50mm3, mice were randomized into two groups (N = 5 in each group). The first group was treated with PBS, once daily, using an intraperitoneal injection. The second group was treated with 50 mg/kg quercetin, once daily with an intraperitoneal injection. Tumor size and weight are measured every three days. After 21 days post-inoculation, the nude mice were sacrificed and tissue was removed for further experiments.
2.10. Statistical analysis.
All data is presented as mean ± SD, and analyzed using SPSS (v.20.0; IBM Corp.). Differences between multiple groups were analyzed using one-way ANOVA followed by Dunnett's post hoc test. Furthermore, a P-value of < 0.05 was considered statistically significant.