Cell lines and culture conditions
Two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and BT-549, and one hormone-receptor positive breast cancer (HRBC) cell lines, MCF-7, were purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX, USA), 100 μg/mL streptomycin, and 100 U/mL penicillin (Gibco, Grand Island, NE, USA) at 37°C in a humidified chamber with 5% CO2. The culture medium was replaced every 3 days.
Compounds
Deferoxamine (DFO), an iron-chelating agent, was purchased from Novartis (Basel, Switzerland). Deferasirox, an another iron-chelator, was purchased from Novartis Pharma (Basel, Switzerland). Eribulin was provided by Eisai Co. (Tokyo, Japan).
Cytotoxicity assay
The sensitivity of the three human breast cancer cell lines to two iron chelators was evaluated using a cell proliferation (MTT) assay kit according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells (1 × 103 cells/well in 96-well plates) were cultured for 24 h in 90 μL of DMEM with 10% FBS. A 10-μL aliquot of medium containing the indicated concentration of DFO or deferasirox was then added to each well. After incubation for 72 h, 10 μL of the MTT reagent was added to each well; 2 h later, the medium was discarded and replaced by 100 μL of dimethyl sulfoxide. After shaking the plates for 10 min, the absorbance of each well was measured at 510 nm using a microplate reader (Perkin-Elmer, Waltham, MA, USA). All samples were analyzed at least three times. [15]
Wound-healing assay
MDA-MB-231 cells were cultured in 96-well plates. After the cells reached 80–90% confluence, a wound was created in the cell monolayer using the WoundMaker tool (Essen Bioscience, Ann Arbor, MI, USA). The cells were cultured in DMEM with 1% FBS and 10 or 100 μM DFO for 30 h. Scratched fields were photographed every 2 h using an Incucyte Live-Cell Imaging System (Essen Bioscience). The degree of cell migration was calculated as 100 × the wound closure area at each time point/the wound area at time 0 [16].
Analysis of cell cycle progression
Cells (1 × 106 cells/well) were seeded into six-well tissue culture plates. After 24 h, the cells were harvested and washed twice with phosphate-buffered saline and stained using the CycleTESTTM PLUS DNA Reagent kit according to the manufacturer’s instructions (Becton Dickinson Biosciences, CA-San Jose, USA). Staining intensity was quantified using a BD LSR II flow cytometer with FACSDivaTM software (Becton Dickinson Biosciences) [17, 18].
Quantitative real-time-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells using a RNeasy Mini kit (Qiagen, Valencia, CA, USA). cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). The RT step was performed at 37℃ for 15 min, 50℃ for 5 min, and then 98℃ for 5 min. cDNA was amplified via qRT-PCR using Taq DNA polymerase (Nippon Gene, Tokyo, Japan) and the StepOnePlus RT-PCR system (Applied Biosystems, Foster City, CA, USA). The following TaqMan gene expression assays for used: assay ID, Hs00154208 (CA9); assay ID, Hs00911700 (KDR); assay ID, Hs00983056 (CDH2); assay ID, Hs00232783 (ZEB1); assay ID, Hs01125301 (CD274); and assay ID, Hs02758991 (GAPHD) (Applied Biosystems). RT-PCR was performed at 95°C for 15 s, followed by 40 cycles at 60°C for 60 s [15].
In vivo tumor model
Invivo experiments were conducted on 4-week-old female athymic BALB/c nu/nu mice (CLEA Japan, Tokyo, Japan) using a protocol approved by the Osaka City University Ethical Committee, Osaka, Japan. All studies on mice were conducted in accordance with the National Institute of Health (NIH) ‘Guide for the Care and Use of Laboratory Animals’. The mice were housed in a standard animal laboratory with ad libitum access to food and water. MDA-MB-231 cells (107 cells) were injected into the backs of the mice to produce subcutaneous xenografts. After a week, the mice were randomized into two groups: (1) control (saline alone) and (2) DFO (20 mg/kg/day, 5 days/week). For evaluation of the combination therapy, the mice were randomized into four groups: (1) control (saline alone), (2) DFO alone, (3) eribulin alone (0.8 mg/kg/day, 5 days/week), and (4) DFO plus eribulin.
Both DFO and eribulin were dissolved in DMEM without FBS. DFO was directly injected into the tumor, and eribulin was intravenously administered. The volumes (length × width) of the resultant tumors were measured weekly. Animals were sacrificed and autopsied at 6 weeks after cell inoculation [17].
Statistical analysis
Statistical analyses were performed using JMP13 software (SAS Institute, Cary, NC, USA). Comparisons between groups were made using Student’s t-test. A p value <0.05 was considered to indicate statistical significance.