Gadolinium-Based AGuIX Nanoparticles.
The synthesis and material characterization of the AGuIX nanoparticles was performed as previously described.
Two human breast cancer cell lines (MDA-MB-231 and MDA-MB-468) were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and cultured in an incubator at 37 ℃ with a humidified atmosphere of 5% CO2. All cells were purchased from the American Type Culture Collection (Teddington, UK).
Therapy study in mice
All animal experiments were licensed by the relevant national and local authorities (Science and Technology Bureau in Tianjin). Female nude mice (HfK Bioscience Co., Ltd., Beijing, China), aged 4 to 5 weeks and weighing approximately 16 g each, were subcutaneously injected in the thigh with 1 × 107 MDA-MB-231 cells. The in vivo experiments were performed when the tumor volume reached 4 × 5 mm3. The mice were randomly divided into four groups, with at least four mice per group, according to whether AGuIX nanoparticles were added to the experimental group and whether the mice were irradiated. Each mouse received a single intravenous injection of 420 mg/kg of AGuIX nanoparticles 30 min prior to local irradiation (10 Gy). Tumor volumes were measured every other day until the mice were euthanized. The experimental samples were collected at the corresponding times.
Histology and immunohistochemistry
The mice were treated as described above in the “therapy study in mice” section. The tumor, liver, spleen, lung, and kidney were harvested 24 h after irradiation and fixed in 10% formalin followed by paraffin embedding for 4-hydroxynonenal, Ki67, and PCNA (Cell Signaling Technology [CST], Boston, MA, USA) staining. The secondary antibody used was PV-9000 (Zsbio Commerce Store, Beijing, China). The images were obtained using an Olympus microscope (BX63, Olympus Life Science, Beijing, China). The ratio of positive nuclear area to total nuclear area for the tumors and the ratio of positive nuclei to total nuclei for the healthy tissues determined the tissue proliferation and lipid oxidation levels, respectively, in each group.
Cell viability assay
The MDA-MB-231 and MDA-MB-468 breast cancer cells in the logarithmic phase were seeded into 96-well plates (3 × 103 cells per well). The concentration gradients were 0 mM, 0.5 mM, 0.8 mM, 1 mM, and 2 mM. A common medium was added around the experimental well to prevent evaporation to reduce error. The completely adherent cells were then incubated with 1 mM of AGuIX nanoparticles (Gd3+) at 37 ℃ for 1 h prior to irradiation with a radiation dose of 2Gy, 4Gy, or 8Gy. The medium was replaced with 100 µL of fresh medium containing 10 µL CCK-8 reagent (Beyotime Biotechnology, C0043, Shanghai, China). After incubation for 2 h in a humidified incubator (37 ℃, 5% CO2), the absorbance was measured at 450 nm using a Bio-Rad microplate reader.
Clonogenic survival assay
MDA-MB-231 and MDA-MB-468 cells in the logarithmic phase were seeded into 6-well plates at a density of 1,000 cells per well and cultured for 24 h. The completely adherent cells were then incubated with 1 mM of AGuIX nanoparticles (Gd molar equivalent concentration) at 37 ℃ for 1 h prior to irradiation with a radiation dose of 1 Gy, 2 Gy, or 4 Gy. After incubation for 2 weeks, the cells were stained with 0.5% crystal violet. Colonies of more than 50 cells were counted in each well. The surviving fraction was calculated using GraphPad Prism 8 and normalized to that of the unirradiated control cells. The dose survival curve was plotted using the multi-target single-hit model (y = 1-(1-exp(-k*x))^N).
ROS and lipid peroxidation assays
The MDA-MB-231 and MDA-MB-468 cells were seeded into 6-well plates (1–2 × 105 cells per well) 24 h prior to irradiation, and they were pretreated with or without AGuIX nanoparticles for 1 h. After the cells were incubated for 24 h or 48 h, fresh medium containing either ROS assay reagent (ThermoFisher, 88-5930-74) for ROS measurements or BODIPY 581/591 C11 dye (ThermoFisher, D3861) for lipid peroxidation measurements was added to each well, according to the manufacturer’s instructions. After incubation at 37 ℃ for 30 min, the cells were washed with phosphate-buffered saline and trypsinized to obtain a cell suspension. ROS and lipid peroxidation levels were analyzed by flow cytometry (BriCyte E6, Mindray, Shenzhen, China).
The MDA-MB-231 and MDA-MB-468 cells were incubated at 37 ℃ for 1 h with AGuIX nanoparticles (1 mM), and then the cells were irradiated at a dose of 8Gy. The cell suspension was collected at 30 min after irradiation and 1 h after irradiation. The cell suspension was mixed with 0.75% low melting point agarose gel (Promega, Madison, WI, USA), dropped onto a glass slide containing 0.75% normal melting point agarose gel (Biowest, Niuele, France), and then spread evenly. The slides were placed in alkaline lysis solution (pH 10) for cell lysis at 4 ℃ for 2.5 h. After the lysis was complete, the slides were transferred to a horizontal electric pool with TBE buffer for DNA dissociation and electrophoresis. Next, the cells on the slides were neutralized and stained with ethidium bromide, then observed and photographed with a fluorescence microscope (Eclipse 90i, Nikon, Tokyo, Japan). At least 200 cells in each group were analyzed with CASP software (Wroclaw, Poland).
The MDA-MB-231 and MDA-MB-468 cells were irradiated with 4 Gy and treated with or without AGuIX nanoparticles (1 mM). Then, 2 h or 24 h after irradiation, the cells were fixed with 4% paraformaldehyde for 15 min, then 0.3% TritonX-100 (prepared with 1% BSA in phosphate-buffered saline) was used to break the membrane for 20 min, then phosphate-buffered saline was used to wash the cells three times, and finally 1% BSA was used to seal the cells for 1 h. The cells were incubated with Phospho-Histone H2AX (Ser139) primary antibodies (Millipore, Belford, MA, USA) overnight at 4 ℃ according to the proportions noted in the instructions. The next day, the cells were incubated with the secondary antibody at room temperature for 2 h. Finally, anti-quenching DAPI (Vector, Burlingame, CA, USA) was added, and the cells were observed and photographed using the EVOS inverted fluorescence microscope (ThermoFisher, Waltham, MA, USA). Cells with more than 10 foci were identified as positive cells.
Cell cycle analysis
The MDA-MB-231 and MDA-MB-468 cells were collected and fixed in precooled 70% ethanol at 6 h, 12 h, or 24 h and then stored at -20 ℃. Propidium iodide staining solution (Solarbio, Beijing, China) was added to each tube of cells, and the cells were bathed in this solution for 15 min at 37 ℃. Cells were detected by flow cytometry (BriCyte E6, Mindray, Shenzhen, China), and the percentage of cells in each phase of the cell cycle was calculated by FlowJo software (FlowJo 7.6, Treestar, USA).
The MDA-MB-231 and MDA-MB-468 cells were collected, and the supernatant was removed by centrifugation. Then, 1 × Binding Buffer from the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, San Diego, CA, USA) was added to the cells. Next, 5 µL of the FITC dye solution and propidium iodide dye solution were added to the corresponding tube, which was then placed away from light for 15 min at room temperature. The results were measured and analyzed by flow cytometry (BriCyte E6, Mindray, Shenzhen, China).
Western blot analysis
The MDA-MB-231 cells were irradiated at a dose of 8Gy and treated with or without AGuIX nanoparticles (1 mM). Then, the protein was extracted from the treated cells at 30 min, 2 h, 6 h, or other times. The western blot was performed using the following primary antibodies: NRF-2 (Proteintech, 16396-1-AP), cleaved caspase-3 (CST, #9664), PARP (CST, #9532), MRE11 (CST, #4895), RAD50 (CST, #3427), (p)NBS1 (CST, #3001), (p)ATM (CST, #92356), (p)CHK2 (CST, #2197), (p)p53 (CST, #82530), (p)BRCA1 (CST, #9010), KU70 (CST, #4588), KU80 (CST, #2753), GPX4 (Abcam, ab125066), ACSL4 (Abcam, ab155282), SLC7A11/xCT (Abcam, ab37185), TFR (Abcam, ab84036), and FTH1 (Abcam, ab65080). A digital imaging system (Bio-Rad, Hercules, CA, USA) was used for imaging and photographing.
Quantitative real-time polymerase chain reaction
The MDA-MB-231 cells were collected 24 h after irradiation, and RNA was extracted with Trizol® (Ambion, Life Technologies, Austin, TX, USA). The mRNA expression levels were detected, and the program was set according to the manufacturer’s standard protocol. The threshold cycle (Ct) values for each gene were normalized to those of GAPDH, and the 2−ΔΔCt method was used for quantitative analysis. Complementary DNA from cell samples was amplified with specific primers. The primers were as follows: hGAPDH-F, GGAGCGAGATCCCTCCAAAAT; hGAPDH-R, GGCTGTTGTCATACTTCTCATGG; hNRF2-F, CACATCCAGTCAGAAACCAGTGG-3
hNRF2-R, GGAATGTCTGCGCCAAAAGCTG-3. hGPX4-F, GAGGCAAGACCGAAGTAAACTAC; hGPX4-R, CCGAACTGGTTACACGGGAA; hSLC7A11-F, TCATTGGAGCAGGAATCTTCA; and hSLC7A11-R, TTCAGCATAAGACAAAGCTCCA.48
The pretreatment of the cells was the same as that noted above for the ROS detection experiment. The cells were removed 24 h after irradiation. The buffer containing the test compound and/or vehicle was removed from the cells and discarded. Next, 50 µL of Total Glutathione Lysis Reagent or Oxidized Glutathione Lysis Reagent (Promega, Madison, WI, USA) was added to each well. Then, 50 µL of Luciferin Generation Reagent was added to each well, and incubation at room temperature was carried out for 30 min. Subsequently, 100 µL of Luciferin Detection Reagent was added to each well. The plate was shaken briefly, and then luminescence was measured after waiting 15 min. Finally, the glutathione/glutathione disulfide ratio was calculated according to the measured data and the standard curve.
GPX4 activity assay
The MDA-MB-231 and MDA-MB-468 cells were seeded into 6-well plates (1–2 × 106 cells per well) 24 h prior to irradiation, and they were pretreated with or without AGuIX nanoparticles for 1 h. Continue to incubate the cells for 24 hours, collect and wash the cells, and then detect and analyze the GPX4 activity of each treatment group according to the experimental steps provided by the kit(Abcam, ab102530).
MDA-MB-231 cells were plated in 6-well plates and transfected with NRF2, SLC7A11 and GPX4 siRNA for 48 h by Refect reagent (Baidai, China). Knockdown efficiency was verified by extracting cell RNA for PCR or cell lysate for western blot. The all siRNA and the negative control siRNA were synthesized and purified by GenePharma (Shanghai, China). The sequences of the used siRNAs are listed below. NRF2-homo-sense:5’-CCGGCAUUUCACUAAACACAA-3’; SLC7A11-homo-sense: 5’-CCAGAUAUGCAUCGUCCUUTT-3’;GPX4-homo-sense:5’-GGAGUAACGAAGAGAUCAATT-3’.
Quantification and statistical analyses
GraphPad Prism 8.0 and SPSS 19.0 software were used for the statistical analysis of the experimental data. Data were presented as means ± standard deviations. Statistical significance (P-values) was calculated using independent sample t-tests. GraphPad Prism 8.0 and Image Lab software were used for drawing and image processing.