Cell culture condition and establishment of a sorafenib-resistant cell line, BaF3/ITD-R
The mouse BaF3 hematopoietic progenitor cell line transfected with FLT3/ITD (BaF3/ITD) was kindly provided by Dr. Donald Small (Johns Hopkins University, Baltimore, MD, USA).The human leukemia cell line, MV4-11, expressing the FLT3/ITD mutation was obtained from the American Type Culture Collection (Manassas, VA, USA). Generation of the sorafenib-resistant cell lines (BaF3/ITD-R, MV4-11-R) and the culture conditions used were both performed as previously described .
Global untargeted metabolomics with liquid chromatography–mass spectrometry (LC–MS)
An equal number of BaF3/ITD and BaF3/ITD-R cells (~ 5×106 cells) in the exponential growth phase were collected in 1.5-mL Eppendorf tubes and rinsed twice with 1 mL of ice-cold normal saline solution. Metabolite extraction and analysis with ultra-high-performance liquid chromatography electrospray ionization mass spectrometry (UHPLC-ESI–MS) analysis (Q Exactive; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was performed as follows. One mL of ice-cold MilliQ water (Millipore, Burlington, MA, US) was added and the cell suspensions were lysed by two freeze–thaw cycle (frozen in liquid nitrogen and thawed at 37 °C for 10 min), followed by 30 s of sonication on ice. Next, 900 μL pre-chilled methanol (− 20 °C) was added to 300 μL cell suspension. The mixture was vigorously vortexed and centrifuged at 14,000×g for 15 min at 4 °C to precipitate proteins and particulates. The supernatant containing the polar extracts was transferred to a 1.5 mL Eppendorf (Hauppauge, NY, USA) and evaporated overnight. Five biological replicates were prepared for ultrahigh-performance liquid chromatography electrospray ionization mass spectrometry (UHPLC-ESI–MS) analysis (Q Exactive, Thermo Fisher Scientific). Polar metabolites were separated on a HILIC (Hydrophilic interaction chromatography) Silica column (Waters, Milford, MA, USA) with column temperature at 40 °C using a gradient elution program at a flow rate of 300 μL/min. The samples were cooled in an auto-sampler at 10 °C and the injection volume was 5 μL. Samples were run in both positive and negative ionization mode. Mass spectrometric data of polar metabolites was acquired at full scan mode (70–1050 m/z [mass to charge ratio]).
Total ion chromatograms and mass spectra data were generated using the Thermo Scientific SIEVE software (Thermo Fisher Scientific, Waltham, MA, USA). Peak picking, alignment, deisotoping and integration were performed to produce a list of mass and retention time pairs with corresponding intensities for all detected peaks. A two-tailed Student’s t test was used to detect the difference of metabolite intensities between two samples (A P-value < 0.05 was considered to be significant). Orthogonal partial least squares-discriminant analysis (OPLS-DA) was used to identify the differential metabolites between two groups using the software SIMCA-P version 14, (Umetrics, Umeå, Sweden). The metabolites were then putatively identified by accurate mass [3 ppm (parts per million) mass error] and fragmentation pattern match (MS/MS spectrum). Structural annotation of the metabolites was searched in public databases including the mz cloud (https://www.mzcloud.org/), HMDB (http://www.hmdb.ca/), and METLIN (http://metlin.scripps.edu).
Pentose phosphate flux analysis with gas chromatography/mass spectrometry (GC–MS)
Briefly, the cells were washed with 0.9% saline and quenched with 500 µL of − 20 °C methanol. Then 200 µL of ice-cold water containing 1 µg norvaline was added as internal standard, after which 500 µL of − 20 °C chloroform was then added to the samples, vortexed for 15 min and centrifuged at 14,000×g for 10 min. The top aqueous layer (polar metabolites) were collected and dried with speed vacuum for GC–MS analysis.
For derivatization, dried polar metabolites were dissolved in 20 µL of 2% (w/v, weight/volume) methoxyamine hydrochloride (Sigma-Aldrich) in pyridine and warmed at 37 °C for 60 min. Subsequent conversion to their tert-butyldimethylsilyl (tBDMS) derivatives was accomplished by adding 30 µL N-methyl-N-(tert-butyl-dimethylsilyl) trifluoroacetamide and 1% tert-butyldimethylchlorosilane (Regis Technologies) and incubating at 37 °C for 30 min. After centrifugation, the top aqueous layer was collected (~45 µL) and mixed with 55 µL pyridine in 150 µL glass inner tube (Waters).
GC–MS analysis was performed using the Thermo 1300 with a 30-m TG-35MS capillary column (Agilent Technologies) connected to Thermo ISQ QD MS. Electron impact ionization (EI) mode was selected and ionization energy was 70 eV (electronvolt). In splitless mode, 1 µL of the derivatized sample was injected at 270 °C, using helium as the carrier gas at a flow rate of 1.5 mL/min. For analysis of organic and amino acid derivatives, the initial temperature of the GC oven was held at 100 °C for 2 min followed by an increase to 255 °C at a rate of 3.5 °C/min and then ramped to 320 °C at 15 °C/min for a total run time of approximately 50 min. The MS source was held at 300 °C, and the detector was operated in scanning mode, recording ion abundance in the range of 100–650 m/z.
Determination of cellular NADP+/NADPH and GSH levels
Nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) levels were analyzed using an NADP/NADPH Quantification Colorimetric Kit (BioVision, Milpitas, CA, USA), and the total cellular glutathione (GSH) and oxidized glutathione (GSSG) were measured using a Glutathione Fluorometric Assay Kit (BioVision, Milpitas, CA, USA). NADP and NADPH levels were analyzed using NADP/NADPH Quantification Colorimetric Kit (Biovision, Milpitas, CA). A total of 4 × 106 cells were washed with cold PBS and lysed with NADP/NADPH extraction buffer. The supernatant was collected for measurement of NADPH and NADP+/NADPH by UV spectrophotometer at 450 nm. Total Cellular glutathione (GSH) and oxidized glutathione (GSSG) was measured using Glutathione Fluorometric Assay Kit (Biovision, Milpitas, CA, USA). Then, 0.4 × 106 cells were prepared by deproteination and subsequent reagents were added following the manufacturer’s instructions. The fluorescence intensity of samples and standards was measured at excitation/emission (Ex/Em) wavelengths = 340/420 nm. Cellular GSH and GSSG contents were calculated with the standard curve generated in parallel experiments and normalized to cell counts.
Quantitative real-time PCR
Total RNA were extracted by Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using the PrimerScript RT reagent kit (TaKaRa, Dalian, China). Quantitative real-time PCR was performed using SYBR® Premix Ex Taq™ II (Clontech) and CFX96 real-time PCR detection system (Bio-Rad). Primers used for BaF3/ITD or BaF3/ITD-R cells were GAPDH (F: 5′-TGGATTTGGACGCATTGGTC-3′, R: 5′-TTTGCACTGGTACGTGTTGAT-3′), G6pdx (F: 5′-CACAGTGGACGACATCCGAAA-3′, R: 5′-AGCTACATAGGAATTACGGGCAA-3′), H6pd (F: 5′-AAGATGCTCCTAGCGGCAATG-3′, R: 5′-TCCAGGTATAGCTGAAACAGTCC-3′), Prps1 (F: 5′-ACTTATCCCAGAAAATCGCTGAC-3′, R: 5′-CCACACCCACTTTGAACAATGTA-3′), Prps2 (F: 5′-ATGCCTAACATCGTGCTCTTC-3′, R: 5′-GATCTCGACACTGGTCTCCTG-3′); Rbks(F: 5′-GGTTCCTGCATGACCGACC-3′, R:5′-TGCCAAAAGAATCGTTGCCAA-3′), Rpe (F: 5′-GCACCTGGATGTAATGGACGG-3′, R: 5′-CCTGGCCTAGCTGCTTTCG-3′), Rpia (F: 5′-AAGGCCGAGGAGGCTAAGAA-3′, R: 5′-CTTTCAGCTATTCGCTGCACA-3′), Taldo1 (F: 5′-GTAAAGCGCCAGAGGATGGAG-3′, R: 5′-CTCTTGGTAGGCAGGCATCT-3′); Tkt (F: 5′-ATGGAAGGTTACCATAAGCCAGA-3′, R: 5′-TGCAGCATGATGTGGGGTG-3′). Primers used for MV4-11 or MV4-11-R cells were Actin (F: 5′-CATGTACGTTGCTATCCAGGC-3′, R: 5′-CTCCTTAATGTCACGCACGAT-3′), G6PD (F: 5′-CGAGGCCGTCACCAAGAAC-3′, R: 5′-GTAGTGGTCGATGCGGTAGA-3′), H6PD (F: 5′-GCAGAGCACAAGGATCAGTTC-3′, R: 5′-GGCAGCTACTGTTGATGTTGC-3′), PGLS (F; 5′-GGAGCCTCGTCTCGATGCTA-3′, R: 5′-GAGAGAAGATGCGTCCGGT-3′) PRPS1 (F; 5′- ATCTTCTCCGGTCCTGCTATT-3′, R: 5′-TGGTGACTACTACTGCCTCAAA-3′), PRPS2 (F: 5′-AGCTCGCATCAGGACCTGT-3′, R: 5′- ACGCTTTCACCAATCTCCACG-3′); RBKS (F: 5′-ATGGTCTGCCAGCTCGAAATA-3′, R: 5′-GAGAGGGTGTAGAACTGGGGA-3′),
RPE (F: 5′- CAGGAGCCAATCAGTACACCT-3′, R: 5′-CAAGGCCAACCTGCAATGG-3′), RPIA (F: 5′-AGTGCTGGGAATTGGAAGTGG-3′, R: 5′-GGGAATACAGACGAGGTTCAGA-3′), TALDO1 (F: 5′-CAGCACAGATGCCCGCTTA-3′, R: 5′-CGGCCCGGAATCTTCTTTAGTA-3′); TKT (F: 5′-TCCACACCATGCGCTACAAG-3′, R: 5′-CAAGTCGGAGCTGATCTTCCT-3′).
Western blot analysis
Collected cells were homogenized in lysis buffer (5% SDS, 10 mM EDTA, 50 mM NaCl, 10 mM Tris–HCl). Protein concentrations were determined using pierce BCA protein assay (Thermo Fisher, Rockford, IL, USA). Proteins were resolved using SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Membranes were blocked in 5% milk, incubated with primary antibody at a concentration of 1:1000, then incubated with secondary antibody at a concentration of 1:10,000 and read using ECL regent (Bio-Rad, Richmond, CA, USA). Antibodies anti-GCLM, anti-GSS, anti-GCLC, anti-GSR, anti-GPX1, anti-SOD1, anti-SOD2, anti-TKT, anti-G6PD, anti-CGL and anti-Actin were purchased from Abcam. Anti-NRF2 and anti-CBS antibody was purchased from Cell Signaling Technology.
Cell viability inhibition was determined by MTS assay. Cells were seeded in a 96-well plate with indicated agents for 72 h. Overall, 20 μL MTS reagent (Promega, Madison, WI, USA) was added to each well and incubated for an additional 4 h at 37 °C. The optical density (OD) was determined by a microplate reader (Thermo, Helsinki, Finland) at 490 nm.
Cell proliferation assay
Cell proliferation was measured by direct cell counting. The cells were seeded in 12-well plates and cultured in the indicated media. The number of cells was counted every 24 h for up to 72 h using a Coulter Z2 Particle Counter & Size analyzer.
Measurement of intracellular cysteine
Intracellular cysteine levels were measured as previously reported . Cells were harvested and washed with ice-cold PBS, and then sulfosalicylic acid (SSA) was added to each sample. After centrifugation, the supernatant was collected. The pH of supernatant was adjusted to 8.3 using NaOH. Samples were reduced with 5 mM dithiothreitol (DTT) for 15 min at room temperature. Following reduction, samples were acidified with glacial acetic and then reacted with acid ninhydrin reagent for 10 min at 100 °C. The samples were cooled and diluted with 95% ethanol. The absorbance of samples was measured at 560 nm, and cysteine levels were quantified using cysteine hydrochloride standards (0–500 µM) processed in the same manner as the samples.
Ingenuity pathway analysis (IPA) of differential metabolites
PubChem compound identification number (CIDs) and corresponding fold changes (≥ 1.2 or ≤ 0.80) of differentially involved metabolites between BaF3/ITD and BaF3/ITD-R cells were uploaded to the IPA platform (http://www.ingenuity.com; QIAGEN, Redwood City, CA, USA) in order to identify the relevant biological functions that were most significant in the uploaded datasets.
Analysis of extracellular fluxes
Extracellular fluxes, including in glucose/glutamine uptake and lactate/glutamate secretion, were measured using a Yellow Springs Instrument 2950D-1 (YSI) Biochemistry Analyzer (YSI Inc, Yellow Springs, Ohio, USA). The quantities (pmol/cell/h) were determined by the difference in substrate concentrations between the final spent medium and the initial medium (RPMI 1640 medium with 10% FBS, GIBCO, Waltham, MA, USA), and normalized by the cell number over 24 h .
Pentose phosphate flux analysis with gas chromatography/mass spectrometry (GC–MS)
A total of 1 × 106 BaF3/ITD or BaF3/ITD-R cells were cultured in a glucose-free RPMI-1640 medium (Gibco, Waltham, MA, USA), supplemented with 11.1 mmol/L [1,2-13C2]-glucose (Cambridge Isotope Laboratories, Tewksbury, MA, USA) and 10% fetal bovine serum (FBS) in 6-well plates for 24 h. Three biological replicates were prepared. Intracellular metabolites were extracted using a methanol/water/chloroform method as previously described . The extraction, derivatization, and GC–MS analysis procedures were performed as described in above “Pentose phosphate flux analysis with gas chromatography/mass spectrometry” section. Glucose flux through the PPP was calculated as the extracellular glucose uptake flux multiplied by the ratio of (M + 1)/[(M + 1) + (M + 2)], where (M + 1) and (M + 2) are two isotopologues of pyruvate derived from [1,2-13C2]-glucose.
Apoptosis and cell cycle assays
The collected cells for apoptosis were stained with Annexin V-FITC and propidium iodide (KeyGEN, Nanjing, China) in binding buffer according to the company’s manual instruction. Apoptosis was determined using FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA). For cell cycle analysis, cells were serum starved overnight and stimulated with complete medium for 6 h. Cells were fixed in 70% ethanol, stained with propidium iodide and measured by flow cytometer.
The bars indicate the mean ± standard error of the mean (S.E.M.). The differences between two groups of data were evaluated using the Student’s t test by Prism GraphPad (San Diego, CA, USA). A P value < 0.05 was considered as statistically significant.