Drugs and Reagents
Tan IIA (Purity: 99.35%, Fig. 1) was purchased from Chengdu Mansite Biotech Co. Ltd (China, Lot. MUST-19090910). Doxorubicin was obtained from Beijing Solarbio Science & Technology Co., Ltd. (China, Lot. 730M024). Cell Counting Kit-8 was purchased from MedChemExpress (USA, Lot. #67666). Matrigel matrix was purchased from Corning (USA, Lot. 9266008). Antibodies against Bcl-2 (ab79849), NF-κB1(ab32360), P53 (ab26), phospho-P53(ab33889) and β-actin (ab8226) were purchased from from Abcam (UK).
Cell Lines And Cultures
Murine triple negative breast cancer 4T1 cell lines were from Tianjin Medical University Cancer Institute and Hospital provided. The Cells were maintained in RPMI-1640 supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 mg/mL) in a humidified atmosphere of 50 mg/mL CO2 at 37°C.
Cell Proliferation Assays
CCK-8 viability assay was performed to evaluate the effects of TAN IIA on cell proliferation. For this, 4T1 cells were suspended with 0.25% trypsin and inoculated into 96-well plates at a density of 3 × 103 cells per well. 4T1 cells were treated with TAN IIA at concentrations of 10, 20, 30, 40, 50, 60, 70 or 80 µM for 24, 48, and 72 hours, respectively. Then the cells were incubated with 10 µl CCK-8 solution for 60 min. Amount of viable cells was estimated according to absorbance (Abs) at 450 nm using a microplate reader (IMark, Bio-Rad). The cell inhibition rate was calculated as the following: inhibition rate (%) = ((Abs control group − Abs experiment group) / (Abs control group − Abs blank group)) × 100% . The SPSS 18.0 software was adopted to calculate 50% inhibitory concentration (IC50) value. All experiments were performed in triplicate and repeated three times.
Colony Formation Assays
Transfected cells were reseeded into six-well plate with 500 cells per well by treating with TAN IIA for 48 hours. Cells were further treated with 0 (blank), 12.5, 25, or 50 µM TAN IIA. After approximately 12 days of cultivation at 37°C, the natural colonies were washed with 1 × PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Then the colonies were stained with 0.5% crystal violet for approximately 20 min. Finally, the stained colonies were imaged and quantified. Colonies formed with over 50 cells were calculated and recorded for statistical analysis. All experiments were performed in triplicate and repeated three times.
Network Pharmacology Analysis
As in our previous researches [15–16], the possible targets of Tan IIA were predicted from the following two databased:①BATMAN-TCM  (http://bionet.ncpsb.org.cn/batman-tcm/); ②TCMSP Database and Analysis Platform  (https://tcmspw.com/tcmsp.php). Candidate TNBC targets were obtained from CTD  (http://ctdbase.org/) and TTD databases  (http://db.idrblab.net/ttd/). The functions of predicted targets were analyzed using online enrichment analysis . Protein–protein interactions (PPI) was constructed with Cytoscape based on the STRING database .
Docking Exercises Of Ingredients Binding To Main Targets
The crystal structure of p53 was obtained from the Protein Data Bank (PDB ID: 3D06) . The crystal structure of Bcl-2 was also obtained from the Protein Data Bank (PDB ID: 6GL8) . The crystal structure of NF-κB1 was also obtained from the Protein Data Bank (PDB ID: 1MDI) . Other targets’ crystal structures from Protein Data Bank include EGFR (5UG9), CASP3 (4JJE), KIT (3G0E), AKT2 (1O6L) and Bcl-2l1 (3SP7) [26–30]. The docking exercise was conducted through AutoDock 4.2 .
After cell lysis, protein concentration was determined using a BCA protein quantitative kit. Protein lysates were resolved by SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. Thereafter, membrane was blocked with 5% w/v nonfat dry milk in 1xTBST for 2 hours and then incubated with respective primary antibodies (1:1000 dilution) overnight at 4°C. Primary antibodies against the following proteins were used: BCL2, NFκB1, TP53, phospho-TP53 and β-actin (Abcam, Cambridge, UK). Afterward, the membrane was washed 2–3 times with 1xTBST and then incubated with the respective secondary antibody (goat anti-rabbit horseradish peroxidase (HRP) conjugated antibody, 1 : 5,000 dilution) (Abcam, Cambridge, UK) for 1 hour at room temperature. After addition of HRP substrate, membranes were examined using an Image acquisition and analysis system (CHEMIDOC XRS+, Bio-Rad, USA). The band signal of each target protein was quantified by ImageJ and normalized according to respective β-actin levels .
In Vivo Xenografts
2× 106 4T1 cells were injected subcutaneously into the upper middle groin of 5 BALB/c female mice. Tumor volumes were determined every day (Tumor volume = a*b2/2, a and b are the long and short diameters of the tumor, respectively). When the average length of the subcutaneous tumor reached 10 mm, the mice were sacrificed in a humane manner, and then the tumors were collected. The collected tumors were cut into small pieces with a length of 2 mm in the clean area and planted in the fat pads of the fourth pair of left breasts of the anesthetized (10% chloral hydrate) BALB/c female mice. Three days later, mice were randomly divided into 3 groups (n = 18): control group (0.9% saline); Tan IIA group (10 mg/kg Tan IIA); Dox group (2 mg/kg Dox). Drugs were given by intraperitoneal injection every other day, tumor volumes were determined every day. Starting from the 8th day, three mice were randomly selected from each group every 3 days and sacrificed in a humane manner. The tumors of the mice were isolated and stored at low temperature. All experiments were approved by the Experimental Animal Ethics Committee of Nankai University.
Apoptosis in tumor tissue was detected with the In Situ Cell Death Detection Kit (Roche Diagnostic Mannheim, Germany). Briefly, after fixing and permeabilization, the tissue was incubated with the TUNEL reaction mixture. Then was covered with mounting medium containing DAPI. Representative images were acquired using a confocal microscope (Olympus) .
Graphs were generated using Excel, PowerPoint software program, GraphPad Prism 5 and ggplot2.
Statistical significance was determined using the Statistical Package for the Social Sciences (SPSS) 12.0. All experimental data were analyzed by one-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons, and there was no significant difference in the variance homogeneity test (P > 0.05).