Chemicals and reagents
Sodium selenite (Se), methylseleninic acid (MSeA) dihydroethidium (DHE), 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), glutamine (Gln), glucose and manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). BAY-876, deferoxamine mesylate (DFO) and ferrostatin-1 (Fer-1) were purchased from MedChem Express (Monmouth Junction, NJ, U.S.A.). Monosodium glutamate (MSG), salicylazosulfapyridine (SAS) and 2-deoxy-D-glcose (2-DG) were purchased from Aladdin (Shanghai, China). NADPH were purchased from solarbio (Beijing, China). FerroOrange and Liperfluo were purchased from DOJINDO (Kyushu, Japan). Antibodies specific for SLC7A11 (12691), SLC7A11 (98051) and β-actin (3700) were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). The secondary antibodies: Horseradish peroxidase-linked Goat Anti-Rabbit IgG and Horseradish peroxidase-linked Goat Anti-Mouse IgG were obtained from MBL International Corporation (Beijing, China). SLC7A11 small interfering RNA (siRNA) and nontargeting siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture and treatments
HCT116 human colon cancer cells, LLC lung cancer cells, HepG2 liver cancer cells, MCF-7 and MDA-MB-231 breast cancer cells, HK2 normal kidney cells and MRC-5 normal lung cells were grown in Dulbecco’ modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 2 mM L-glutamine unless otherwise indicated. A549 lung cancer cells, DU145 prostate cancer cells were grown in RPMI medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine unless otherwise indicated. For starvation experiments, cells were washed three times with PBS pH 7.2 and then incubated in the indicated starvation conditions. All cultures were maintained in a humidified tissue culture incubator at 37℃ in 5% CO2.
Crystal violet staining
After treatment with agents, the culture medium was aspirated and replaced with 1% glutaraldehyde solution for 15 min. Then, the cells were stained with a 0.02% crystal violet solution for 30 min. After that, the solution was replaced by 70% ethanol for solubilization. The OD value at 570 nm was measured by microplate reader.
Cell death evaluation
Collected cells were incubated with a fluorescein isothiocyanate-labeled annexin V (FITC) and propidium iodide (PI) for flow cytometry analyses. An Annexin V/PI staining kit (MBL International Corporation) was used.
Determination of cell ROS
Reactive oxygen species (ROS) measurement using DHE and DCFH-DA staining was performed as described as follows: cells were trypsinized, washed with PBS, and then resuspended in the medium without FBS and loaded with 10 μM DHE or 20 μM DCFH-DA for 30 min at 37℃, incubated in the dark. Afterward, cells were washed with PBS and resuspended in the PBS used for fluorescence analysis by flow cytometer. Fluorescence increase was estimated utilizing the wavelengths 535 nm (excitation) and 610 nm (emission) for DHE, and wavelengths 485 nm (excitation) and 535 nm (emission) for DCFH-DA.
GSH, NADP+, NADPH and cystetine measurement
Glutathione (GSH) in cell was measured using a commercial kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) following the manufacturer’s instruction. The intracellular levels of NADPH and total NADP (NADPH+NADP+) were measured according to the protocol of manufacturer from DOJINDO (Kyushu, Japan). The quantification of cysteine was determined using Cysteine assay kit (Abcam, Cambridge, United Kingdom).
Cystine measurement by LC-MS
Cells were seeded on 75 cm culture flasks at a density sufficient to ensure approximately 80% confluence at the time of extraction. Cells were trypsinized, washed twice with ice cold PBS, extracted by adding 0.3 mL of an 50% methanol: 50% water mixture (v/v) at ice temperature and then disrupted by ultrasound. Cell debris was pelleted by centrifugation at 12000 rpm for 10 min at 4℃ and the supernatant was transferred to a fresh tube. 20 μL cell extracts, 5 μL internal standard substance (canocinal amino acid mix with 1.23 mM L-Cystine-13C2-15N1, Cambridge Isotope Laboratories) and 40 μL isopropyl alcohol-formic acid（v/v=99:1）were mixtured and vortex oscillated for 2 min, then centrifugated at 12000 rpm for 10 min at 4℃. 10 μL supernatant were derivatized according to the protocol of AccQ Tag kit (Waters, USA) and then analyzed by LC-MS. Analyte concentrations were quantified by comparison to standard curves of cystine prepared by the same method. To determine intracellular concentrations, the concentrations of cell protein in the same volume of cell resuspension were determined.
LC-MS analysis was performed as follows: the LC-MS system consists of a Waters UPLC I-Class system and Waters XEVO TQ-XS quadrupole rods tandem mass spectrometer equipped with a ESI probe. Mass data acquisition and remote control of the LC-MS system were done by Masslynx software. Chromatography was performed with a waters UPLC HSS T3(150 × 2.1 mm, 1.8 μm particle size). The mobile phase consisted of solvent A (0.1 % formic ,water) and solvent B (acetonitrile and water, v/v=95:5) with a gradient elution (0-0.5 min, 96-96% A, 0.5-2.5 min, 96-90% A, 2.5-5 min, 90-72% A, 5-6 min, 72-5% A, 6-7 min, 5-5% A, 7- 7.1 min, 5-96% A, 7.1- 9 min, 96-96% A). The flow rate of the mobile phase was 0.5 mL/min. The column temperature was maintained at 50℃. The injection volume was 5 μL. The exactive was operated in positive ionization mode with an electrospray ionizatio interface. The instrument parameters were as follows: positive ESI source temperature, 50℃, capillary voltage, 1.5 kV, cone voltage, 20 V, cone gas flow, 150L/Hr, desolvation gas flow, 1000 L/Hr.
LPO, LIP measurement
The total cellular labile iron pool (LIP) and lipid peroxidation (LPO) measurement using FerroOrange and Liperfluo were performed as described as follows: cells were trypsinized, washed with PBS, and then resuspended in the PBS and loaded with 1 μM FerroOrange and or 1 μM Liperfluo for 30 min at 37℃, incubated in the dark. Then the cells were analyzed for fluorescence by flow cytometer. Fluorescence increase was estimated utilizing the wavelengths 561 nm (excitation) and 593 nm (emission) for FerroOrange, and the wavelengths 488 nm (excitation) and 535 nm (emission) for Liperfluo.
The cells were transfected with 40 nM SLC7A11 siRNA or nontargeting siRNA using the INTERFERin siRNA transfection reagent according to the manufacturer’s instructions (Polyplus-Transfection, Inc., New York, NY, USA) and then were used for subsequent experiments.
Western blotting was performed according to the method of Yan et al with minor modifications. The cell lysate was prepared in ice-cold radioimmunoprecipitation assay (RIPA) buffer. Cell lysates were resolved by electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA, IPVH00010). The blot was then probed with primary antibody followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies. The signal was visualized by enhanced chemiluminescence (Fisher/Pierce, Rockford, IL, USA, 32106) and recorded on an X-ray film (Eastman Kodak Company, Rochester, NY, USA, XBT-1).
Xenograft tumor models
The animals were housed under specific pathogen-free conditions at 22 ± 2℃ with 55 ± 10% relative humidity and with 12 h day/light cycles. All experiments were performed in accordance with the guidelines established in the Principles of China Agricultural University Institutional Animal Care and Use Committee. For xenograft experiments, 6-8-week-old male C57 BL/6N mice from Charles River (Beijing, China) were subcutaneously injected with 2×106 LLC cells resuspended in 100 μL of PBS, 5 week-old male BALB/c nude mice from Charles River (Beijing, China) were subcutaneously injected into the dorsal side with 3×106 HCT116 cells resuspended in 100 μL of PBS. When tumors were palpable (5 days after inoculation), mice were randomly divided into different experimental groups. Mice were kept on the feeding/fasting protocols performed as described as the reported. In short, fasting cycles were achieved by complete removal of food while allowing free access to water for 24 h from 6 pm to 6 pm of the following day when food was re-supplied ad libitum. Selenite dissolved in water at the dose of 2 mg/kg body weight was administered every 48 h at 9 am (time in fasting cycle) via oral gavage. Body weights were recorded every 2 days, and tumor volumes were measured every 2 days by a digital vernier caliper according to the following equation: tumor volume (mm3) =(length × width2) × 0.5, where the length and width were expressed in millimeters.
Data were presented as the mean±SD. These data were analyzed by ANOVA with appropriate post hoc comparisons among means. P<0.05 was considered statistically significant.