Chemicals and reagents
Eup was kindly provided by Dr. Bo Yang (Zhejiang Chinese Medical University, Hangzhou, China). It was analyzed by NMR and MS. The NMR spectrum of Eup can be seen in supplement materials (Fig. S1). For in vitro research, Eup was dissolved in DMSO at the 50 mM stock solution. For in vivo research, Eup was dissolved in ethanol /Cremophor EL/ saline (5: 5: 90). Cremophor EL, MTT and DAPI were purchased from Sigma-Aldrich. The PI/RNase staining cell cycle kit and Annexin V-FITC/7AAD apoptosis kit were purchased from BD Biosciences (San Diego). BODIPY C11 and phen green SK (PGSK) were obtained from from Thermo Fisher Scientific. Z-VAD-FMK (HY-16658B), Deferoxamine (#HY-B0988), ferrostain-1 (#HY-100579) and glutathione (GSH) (#HY-D0187/CS-7948) were purchased from MCE. Antibodies against Bcl2 (#2870, CST), Bad (#1541, CST), caspase3 (#9662, CST), cleaved caspase 3 (#9664, CST), FTH1 (#4393, CST), p53 (#9282, CST), β-tubulin (#2128, CST), β-actin (#4970, CST), GAPDH (#2118, CST) and horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly). GPX4 (ab125066, Abcam) was purchased from Abcam (Cambridge, MA, USA).
Extraction, isolation and structural identification of Eup
2 Kg Eupatorium cannabinum Linn. coarse powder was soaked in 20-fold 95% EtOH for 2 days, then extracted at room temperature via percolation. The crude extract was added into warm water and suspended to 1.5 L, and isolated with EtOAc (3 × 1.5 L). The EtOAc extract was evaporated. The ethyl acetate-soluble fraction (EEECL) was then subjected to silica gel column chromatography, eluting with a gradient solvent system of PE-EtOAc (20:1 - 0:1 v/v) to obtain 11 fractions (Fr.1~11). Fr. 9 (1.5 g of 19.33 g) was separated by MPLC using MeOH/H2O as the mobile phase (45, 50 and 55%, each 600 mL), flow rate 50 mL/min, to obtained Eup (28.7 mg). The known sesquiterpenoids Eup was identified mainly by comparing NMR spectral data with literature values(23). The structure data can be seen in Fig. 1 a.
The TNBC cells (MDA-MB-231 and MDA-MB-468) cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences. DMEM/F-12 medium, fetal bovine serum (FBS) and trypsin EDTA were purchased from Gibco. Cell lines were maintained in DMEM/F12 medium with 10% FBS and 1% penicillin/ streptomycin at 37 °C in a humidified 5% CO2 and 95% air atmosphere.
In vitro cell viability assay
The cell inhibition rate was evaluated using MTT assay.
In vitro GSH assay
The GSH/GSSG ratios were detected by the GSH/GSSG-Glo Assay kit (Beyotime, China) according to the manufacturer’s suggestion.
Measurement of lipid ROS
The BODIPY C11 (Invitrogen) probe was used to determine lipid ROS according to the manufacturer’s suggestion.
Flow cytometric analysis of cell cycle arrest and apoptosis.
For cell cycle analysis, PI/RNase was used according to the manufacturer’s suggestion. Concerning apoptosis, cells were stained with Annexin-V-FITC/7AAD.
Intracellular chelate iron was detected by the PGSK (Thermo Fisher Scientific), and its fluorescence is quenched by iron.
Mutant p53 knockdown experiments
Short hairpin RNAs (shRNA) were used for packaging into lentivirus (LV). Cells were seeded in a six-well plate and transfected with LV-shRNA targeting mutant p53 (Genechem, China). After transfection, proteins were isolated and analyzed by western blotting.
RNA was obtained by TRIZOL (Invitrogen). Then it was reverse transcribed with PrimeScript™ RT reagent Kit (Takara Bio, Inc.). Afterward, qPCR was performed with SYBR-Green (Bio-Rad Laboratories, Inc.) by the CFX96 Real-Time system (Bio-Rad Laboratories, Inc.). The relative expression levels of mRNA were calculated by 2−∆∆Cq method. The primer sequences are exhibited as follows:
The primer sequences are shown as follows:
Human SAT1 forward: 5′-ACCCGTGGATTGGCAAGTTAT-3′ and reverse，5′-TGCAACCTGGCTTAGATTCTTC- 3′;
Human GAPDH forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse，5′-GGCTGTTGTCATACTTCTCATGG - 3′;
Cell were lysed in RIPA lysis buffer. Then, equal quantity of proteins was separated on SDS-PAGE gels and transferred onto PVDF membranes (Millipore). The membranes were blocked in 5% skimmed milk, and incubated with primary antibodies at 4 °C overnight and probed with appropriate secondary antibodies. Chemiluminescent detection was performed by ECL (Bio-Rad, USA). The primary antibodies used in the study were as follows: anti-Bcl2 (#2870, CST), anti-Bad (#1541, CST), anti-caspase3 (#9662, CST), anti-cleaved caspase 3 (#9664, CST), anti-FTH1 (#4393, CST), anti-GPX4 (ab125066, Abcam), anti-p53 (#2527, CST), anti-β-tubulin (#2128, CST), anti-β-actin (#4970, CST), anti-GAPDH (#2118, CST).
Evaluation of mitochondrial membrane potential (Δψ m)
JC-1 (Beyotime, China) was used to determine Δψ m following the manufacturer’s suggestion.
In vivo tumor model
The experimental procedures of the animal studies were permitted by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University. BALB/c nu/nu female mice (4-week-old; n=6) were purchased from Shanghai Experimental Animal Center (Shanghai, China). MDA-MB-231 cells (2.5×106) were injected subcutaneously into the flank of mice. While tumor volume reaching 50 mm3, the mice were randomly divided into vehicle control(n=6) and Eup group (15 mg/kg, n=6). Drugs were administered by intraperitoneal injection every two days. The animal health and behavior were monitored daily. Tumor growth was measured, and tumor volume was calculated with the following equation: volume = (length ×width2)/2. After 14 days of drug administration, the mice were killed, and tumor tissues were obtained for TUNEL and immunohistochemical experiments.
Tumor tissues were fixed and paraffin-embedded. Sections were dewaxed and rehydrated. After incubation with primary antibody, the slides were then incubated in HRP horseradish peroxidase, stained with DAB (ZSGB-BIO, China), counterstained with hematoxylin.
All data are expressed as the mean ± SD of three independent experiments. Statistical significance was analyzed with a Student's t-test. The criterion of statistical significance was* p<0.05; ** p<0.01; *** p<0.001.