METHODS: qPCR and Western blotting were performed to detect changes in xCT and CD98 expression after glucose withdrawal. Then, the cell viability of OSCCs under the indicated conditions was measured. To identify the GD-responsible transcriptional factors of SLC7A11, we performed a luciferase reporter assay and a ChIP assay. Further, metabolomics was conducted to identify changes in metabolites. Finally, mitochondrial function and ATP production were evaluated using the seahorse assay, and NADP+/NADPH dynamics were measured using a NADP+/NADPH kit.
RESULTS: In OSCCs, system Xc- promoted GD-induced cell death by increasing glutamate consumption, which promoted NADPH exhaustion and TCA blockade. Moreover, GD-induced xCT upregulation was governed by the p-eIF2α/ATF4 axis.
In total 10^6 HSC3 cells were seeded and incubated at 37 °C for 24 h. The next day, cells were treated with high glucose DMEM (Dulbecco's Modified Eagle Medium) (control group; CTR), glucose-free DMEM (glucose deprivation group; G(-)) or glucose-free DMEM with erastin (glucose deprivation with erastin group; G(-)+Era) for 14 h, then cells of each group were trypsined and collected into a 1.5 mL tube. The collected cells were frozen immediately with liquid nitrogen before subsequent experiments.
First 1 mL of methanol:water (4:1, v/v) were added to each sample, then transformed to a 4 mL glass vial. Next 200 μL of chloroform were added to each aliquot, dispersing sample by pipette. Using ultrasonic homogenizer to break up the cells for 3 min at 500 w. All of the mixtures of each sample were transferred to 1.5 mL Eppendorf tubes, 20 μL of L-2- chlorophenylalanine (0.03 mg/mL) dissolved in methanol as internal standard, then extracted by ultrasonication for 20 min in ice-water bath. The extract was centrifuged at 4 °C (13,000 rpm) for 10 min. A total of 400 μL of supernatant in a glass vial was dried in a freeze concentration centrifugal dryer. QC sample was prepared by mixing aliquot of the all samples to be a pooled sample. In total 80 μL of 15 mg/mL methoxylamine hydrochloride in pyridine was subsequently added. The resultant mixture was vortexed vigorously for 2 min and incubated at 37 °C for 90 min. Next 50 μL of BSTFA (with 1% TMCS) and 20 μL n-hexane were added into the mixture, which was vortexed vigorously for 2 min and then derivatized at 70 °C for 60 min. The samples were placed at ambient temperature for 30 min before GC-MS analysis.
The derivatived samples were analyzed on a Trace1310 gas chromatography system coupled to a TSQ 9000 Mass spectrometer with an EI source (Thermo Fisher Scientific, USA). A DB-5MS fused-silica capillary column (30 m x 0.25 mm x 0.25 μm, Agilent J & W Scientific, Folsom, CA, USA) was utilized to separate the derivatives. Helium (> 99.999%) was used as the carrier gas at a constant flow rate of 1.2 mL/min through the column. The injector temperature was maintained at 300 °C. Injection volume was 1 μL. The initial oven temperature was 60 °C, kept for 0.5 min, ramped to 125 °C at a rate of 8 °C/min, to 210 °C at a rate of 8 °C/min, to 270 °C at a rate of 15 °C/min, to 305 °C at a rate of 20 °C/min, and finally held at 305 °C for 5 min.
The derivatived samples were analyzed on a Trace1310 gas chromatography system coupled to a TSQ 9000 Mass spectrometer with an EI source (Thermo Fisher Scientific, USA). The temperature of MS quadrupole and ion source (electron impact) was set to 280 and 330 °C, respectively. Mass data was acquired in a full-scan mode (50-500 m/z) and the solvent delay time was set to 5 min. The QCs were injected at regular intervals throughout the analytical run to provide a set of data from which repeatability can be assessed.
The obtained GC-MS raw data in .raw format were transferred to .abf format via software Analysis Base File Converter for quick retrieval of data. Then, data were imported into software MS-DIAL, which performs peak detection, peak identification, MS2Dec deconvolution, characterization, peak alignment, wave filtering and missing value interpolation. A data matrix was derived. The 3D matrix includes: sample information, the name of the peak of each substance, retention time, retention index, mass-to-charge ratio and signal intensity. In each sample, all peak signal intensities were segmented and normalized according to the internal standards with RSD greater than 0.3 after screening. After the data was normalized, redundancy removal and peak merging were conducted to obtain the data matrix.
The matrix was imported in R to carry out Principle Component Analysis (PCA) to observe the overall distribution among the samples and the stability of the whole analysis process. Orthogonal Partial Least-Squares-Discriminant Analysis (OPLS-DA) and Partial Least-Squares-Discriminant Analysis (PLS-DA) were utilized to distinguish the metabolites that differ between groups. To prevent overfitting, 7-fold cross-validation and 200 Response Permutation Testing (RPT) were used to evaluate the quality of the model. Variable Importance of Projection (VIP) values obtained from the OPLS-DA model were used to rank the overall contribution of each variable to group discrimination. A two-tailed Student’s T-test was further used to verify whether the metabolites of difference between groups were significant. Differential metabolites were selected with VIP values greater than 1.0 and p-values less than 0.05.
Metabolite characterization was based on the LUG database.
Study Protocol URI Study Protocol Version Study Protocol Parameters Name Post Extraction;Derivatization Chromatography Instrument;Autosampler model;Column model;Column type;Guard column Scan polarity;Scan m/z range;Instrument;Ion source;Mass analyzer Study Protocol Parameters Name Term Accession Number ; ;;;; ;;;; Study Protocol Parameters Name Term Source REF ; ;;;; ;;;; Study Protocol Components Name Study Protocol Components Type Study Protocol Components Type Term Accession Number Study Protocol Components Type Term Source REF STUDY CONTACTS Study Person Last Name Wang Study Person First Name Miao Study Person Mid Initials Study Person Email wangm@163.com Study Person Phone +86 15982176890 Study Person Fax Study Person Address Hospital of Huaxi stomatology, the 3rd section, Renmin Nan road, Wuhou, Chengdu, Sichuan, China Study Person Affiliation University of Sichuan Study Person Roles principal investigator role Study Person Roles Term Accession Number http://purl.obolibrary.org/obo/OBI_0000103 Study Person Roles Term Source REF OBI