Cell culture
Human breast cancer cell lines including MCF-7 cells, MDA-MB-231 cells and Adriamycin resistant MCF-7-Adr cells were stored in our laboratory. The MCF-7 and MDA-MB-231 cells were cultured in DMEM medium (KeyGEN, Cat# KGM12800-500) with 10% fetal bovine serum (FBS, BI, Cat# 04-001-1ACS). MCF-7-Adr cells were cultured in 1640 medium (KeyGEN, Cat# KGM31800-500) with 10% fetal bovine serum. All the cell culture contamination was at 37 °C under a humidified atmosphere with 5% CO2.
Reagents
Z-VAD-FMK (APExBIO, Cat# A1902), Necrostatin-1 (APExBIO, Cat# A4213), Ferrostatin-1 (APExBIO, Cat# A4371), NAC (Beyotime, Cat# S0077), CA-074 methyl ester (MCE, Cat# HY-100350), Ammonium ferric citrate (Sigma, Cat# F5879), Deferoxamine mesylate salt (YuanyeBio, Cat# S61301)
Quantitative Real-time PCR (qPCR)
The process of this experiment was referred to our previous study[24].
Western blot
The cells were collected by 1×PBS (phosphate buffer saline) (diluted with double distilled water, Servicebio, Cat# G4207-500ML) and were lysed with RIPA Lysis Buffer (Beyotime, Cat# P0013B) with PMSF (1:100, Beyotime, Cat# ST506) on the ice. After SDS-PAGE (sodium dodecyl sulfate – polyacrylamide gel electrophoresis) analysis, the proteins were transferred on the PVDF (polyvinylidene fluoride) membranes (Millipore, Cat# IPVH00010), which were then incubated with the primary antibodies at 4℃ overnight. The membranes were incubated with the secondary antibodies (Goat Anti-Rabbit IgG (H+L), HRP conjugate; Goat Anti-Mouse, IgG (H+L), HRP conjugate, proteintech, Cat# SA00001-2, Cat# SA00001-1) and protein signals were detected with High-sig ECL Western Blotting Substrate (Tanon, Cat# 180-5001) on Tanon5200. The antibody used in this study including ALDH1A1 (Proteintech,15910-1-AP), OCT3/4 (Wanleibio, Cat# WL01728), GAPDH (Proteintech, Cat# 60004-1-l g), E-cadherin (Proteintech, Cat# 20874-1-AP), N-cadherin (Proteintech, Cat# 22018-1-AP), MMP9 (Affinity, Cat# AF5228), IRP2(Proteintech, Cat# 23829-1-AP), TfR1 (Santa, Cat# SC-32272), ferritin (Abcam, Cat# ab75973).
Flow Cytometry Analysis
MCF-7 and MDA-MB-231 cells were seeded into 6-well plates and treated with different compounds or inhibitors, after 48 h, the cells were stained with Hu CD44 APC (BD Pharmingen, Cat# 559942) and Hu CD24 BV421(BD Pharmingen, Cat# 562789) on the ice in 30 min and then the subpopulation of CD44+/CD24- ratio was analyzed on a MACSQuant flow cytometer (Miltenyi Biotec).
Cell spheroid formation assay
MCF-7 and MDA-MB-231 cells were seeded into low-adherent 24-well plates at a density of 3000 - 5000 cells/well and cultured with the MammoCultTM Human Medium Kit (stemcell, Cat# 05620) supplemented with compounds for 8 days. Then spheroid number was calculated and size was measured under a microscope (Leica).
MTT
Cell viability was measured by MTT (Solarbio, Cat# M8180-250 mg). MCF-7, MDA-MB-231 and MCF-7-Adr cells were seeded into 96-well plates at a density of 3000 - 5000 cells/well. After 24 h, different concentrations of CPUL119, CPUL129 and CPUL 149 were added. 48 h later, 20 μl 5 mg/ml MTT was added into the medium for a 4 h culturing. Then the medium was removed and added 150 μl DMSO into each well. After shocking for 10 min, the absorbance at 490 nm was analyzed on a Biorad iMark.
Detection of Lipid peroxidation
MCF-7 and MDA-MB-231 cells were seeded into 6-well plates. After 24 h, cells were treated with compounds (4.5 μM CPUL119, 1.8 μM CPUL129 and 0.9 μM CPUL149), NAC (5 mM), and DFO (2 μM). 48 h later, the cells were stained with 10 μM Lipid peroxidation sensor (ThermoFisher, Cat# B3932) for 30 min at 37℃. Then lipid peroxidation level was analyzed with a flow cytometer.
Detection of ROS level
MCF-7 and MDA-MB-231 cells were seeded into 6-well plates. After 24 h, cells were treated with compounds (4.5 μM CPUL119, 1.8 μM CPUL129 and 0.9 μM CPUL149). 48 h later, the production of ROS was analyzed by reactive oxygen species assay kit (Beyotime, Cat# S0033S) through a flow cytometer.
GSH determination
The analysis of GSH was performed by Micro Reduced Glutathione (GSH) Assay Kit (Solarbio, Cat# BC1175) following the manufacturer’s recommendation.
Analysis of iron content
The content of iron in the cell was measured by Iron Colorimetric Assay Kit (Applygen, Cat# E1042-100) according to the standard protocol. Briefly, MCF-7 and MDA-MB-231 cells were seeded into 24-well plates. After 24 h, the cells were treated with compounds (4.5 μM CPUL119, 1.8 μM CPUL129 and 0.9 μM CPUL149). 48 h later, the iron concentration was analyzed by Iron Colorimetric Assay Kit.
Wound-Healing assay
MCF-7 and MDA-MB-231 cells were seeded into 6-well plates and cultured in the complete medium until growing up to 90%. Then, a wound was produced on the cell surface, and washed it with PBS twice to remove the cell fragments. Cells were cultured in the medium with low serum concentration(≤2%) contained compounds or solvent. After 24 h and 48 h, the wounds were observed and taken with a microscope. Migration rate = (Dt-D0) / D0, the Dt represents the distance between the wound at different times.
Invasion assay
Cell invasion ability was assayed by transwell invasion analysis. Finally, the Millicell Hanging cell culture inserts (Merck, Cat# MCEP24H48) coated with Matrigel matrix (Corning, Cat# 356234) were prepared in the 24-well plates. The 2 × 105 MCF-7 or MDA-MB-231 cells with 200 μl serum-free medium with different compounds or solvent were added into the upper chamber, and the lower chamber was filled with medium containing 20% serum. After 48 h, the cells were fixed with 70% ethanol for 20 min at room temperature. Then, the invaded cells were stained with crystal violet staining solution (Beyotime, Cat# C0121-100 ml), and observed under a microscope. After then, the chambers were washed with 33% glacial acetic and the absorbance at 570 nm was analyzed on a Biorad iMark, which represents the invaded cell number.
Fluorescence Microscopy Analysis
MCF-7 cells were seeded into glass-bottom culture dishes (NEST, Cat# 801001) to carry on this experiment. After 24 h, these cells were treated with different compounds (4.5 μM CPUL119, 1.8 μM CPUL129 and 0.9 μM CPUL149). 48 h later, the cells were stained with different probes as the following methods. 50 nM LysoTracker Deep Red (ThermoFisher, Cat# L12492) was incubated with cells for 90 min at 37℃. 50 nM MitoTracker Deep Red (ThermoFisher, Cat# M22426) was incubated with cells for 30 min at 37℃. 1 μM FerroOrange (DOJINDO, Cat# F374) was incubated with cells for 30 min at 37℃. 2 μg/ml Hoechst 3342 was incubated with cells for 30 min at 37℃. After stained with different probes, we used a LSM800 to analyze.
RNA sequencing and data analysis
RNA sequencing and data analysis were performed by Novogen (Beijing, China).
Tumorigenesis in vivo
The BALB/c-nude mice (Gempharmatech, Cat# D000521) were female, 3-4 weeks, and were cultured in standard pathogen-free conditions. Each group had six mice. All the experiments were obtained the approval of the Ethics Committee for Animal Experimentation of China Pharmaceutical University. In the tumor-limiting dilution assay, 1×106, 1×105 and 1×104 MCF-7 and MDA-MB-231 cells pre-treated with compounds (4.5 μM CPUL119, 1.8 μM CPUL129 and 0.9 μM CPUL149) for 48 h were implanted in the inguinal mammary gland of mice orthotopically. After 10 days, the mice were sacrificed, and the amounts of tumors in each group were counted. And the stem cell frequencies were analyzed by ELDA (http://bioinf.wehi.edu.au/software/elda/).
Metastasis in vivo
The BALB/c-nude mice (Gempharmatech, Cat# D000521) were female, 3-4 weeks, and were cultured in standard pathogen-free conditions. Each group had five mice. All the experiments were obtained the approval of the Ethics Committee for Animal Experimentation of China Pharmaceutical University. In the metastasis, each mouse was injected with 2×106 MDA-MB-231 cells by intravenous. One week later, these mice were injected with different compounds (10 mg/kg 119, 10 mg/kg 129, 5 mg/kg 129) or solvent (23.7% ethanol, 17%PEG400, 59.3% saline) intravenous every two days. After one month later, the mice were sacrificed. And the lungs were made H&E by Servicebio.
Statistical analysis
The results were presented as mean ± SD, the analysis was Student’s t-test by using GraphPad Prism 5 software. P-values less than 0.05 were considered to be statistically significant. (*p < 0.05, **p < 0.01, ***p < 0.001).