2.1 Cell Culture and Reagents
The human gastric cancer cell lines MGC-803 and HGC-27 were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (C3010-0500, Sartorius, GER) or RPMI-1640 medium (C3010-0500, Sartorius, GER) containing 10% fetal bovine serum (C04001-050, Sartorius, GER) at 37°C in 5% CO2.
MLN4924 was purchased from TagerMol (T6332, Shanghai, China). BAY876 was purchased from Aladdin (B287376 Shanghai, China). Antibodies Cleaved PARP (5625, 1:1000 dilution), Cleaved caspase 9 (52873, 1:800 dilution), Cleaved caspase 3 (9664, 1:800 dilution), Bax (D2E11, 1:1000 dilution), Bcl-2 (15071, 1:1000 dilution), Myt1 (4282, 1:500 dilution), p-wee1(Ser642) (D47G5, 1:500 dilution), p-CDC2 (4539, 1:500 dilution), cyclinB1 (12231, 1:1000 dilution), p21 (2947, 1:600 dilution), Anti-rabbit IgG (7074, 1:10000 dilution), Anti-mouse IgG (7076, 1:10000 dilution) were obtained from Cell Signaling Technology (Boston, MA, USA); anti-γ-H2AX (Ab81299, 1:5000 dilution), anti-GLUT1 (Ab115730, 1:80000 dilution), anti-HIF1 alpha (ab1, 1:1000 dilution) were obtained from Abcam (Cambridge, UK) and antibodies against β-actin (AF0003, 1:1000 dilution) was from Beyotime Biotechnology (Shanghai, China).
2.2 Cell Viability Analysis
Cells in the logarithmic growth phase of MGC-803 and HGC-27 were taken and seeded into a 96-well plate at different cell densities. All cells were then exposed to MLN4924 at different concentrations for 24, 48 or 72 h. After fixing the cells with trichloroacetic acid (TCA), unbound Sulforhodamine B (SRB) (Meilunbio, China) was washed away, and the protein-bound SRB was solubilized and quantified spectrophotometrically, at 560 nm with a full-function microplate detector (BioTek, USA) and calculated cell viability (Cell viability (%) = A560 of the treatment group/A560 of the control group).
2.3 EdU Cell Proliferation Assay
Cell Proliferation was performed using Cell-Light EdU Apollo567 In Vitro Kit (RiboBio, China). Logarithmic growth phase MGC-803 cells (8×104 cells) and HGC-27 cells (5×104 cells) were seeded into confocal dishes. After treated with MLN4924 for 48 h, 100 µM EdU was added and incubated for 2 h. Subsequently, the cells were fixed with 1 mL of 4% paraformaldehyde solution, followed by incubation with Apollo staining at room temperature in the dark for 30 minutes. Then, Hoechst 33342 solution was added and incubated at room temperature in the dark for another 30 minutes. And the samples were ready for imaging under a Confocal laser scanning microscope (Nikon, Japan).
2.4 Cell Cycle Detection
The Cell Cycle Detection Kit (Beyotime, China) was used to detect the cell cycle and cell apoptosis with a flow cytometer (Becton, USA). Propidium Iodide (PI) is a fluorescent dye that binds to double-stranded DNA, producing fluorescence, and the fluorescence intensity is directly proportional to the amount of double-stranded DNA. Based on the distribution of DNA content, cell cycle analysis can be performed. Cells in the logarithmic growth phase of MGC-803 and HGC-27 were taken and seeded into a 6-well plate. After treating the cells with MLN4924 for 48 h, collect and stain them with PI at 37°C in the dark for 30 minutes and started flow detection. The FlowJo-V10 software was used for quantitatively analyzing.
2.5 Cell Apoptosis Analysis
The Annexin V-FITC/PI cell apoptosis detection kit was used to analysis cell apoptosis. FITC-labeled Annexin V as a probe to detect early-stage apoptosis in cells, while PI is used to distinguish between viable early-stage cells and dead or late-stage apoptotic cells. By simultaneously detecting the fluorescence signals of FITC and PI with a flow cytometer (Becton, USA), it allows for quantitative analysis of the proportion of cells in different stages of apoptosis. Cells in the logarithmic growth phase of MGC-803 and HGC-27 were taken and seeded into a 6-well plate. After treating the cells with MLN4924 for 48 h, collect and stain them with Annexin V-FITC and PI staining at 37°C in the dark for 30 minutes. Then started flow detection after diluting with the Binding Buffer. The FlowJo-V10 software was used for quantitatively analyzing.
2.6 Clone Formation Assay
MGC-803 (1500 cells) and HGC-27 (800 cells) cells in the logarithmic growth phase were collected and seeded into a 6-well plate, treated with MLN4924, then cultured at 37°C with 5% CO2. The culture medium was replaced every three days. When visible cell colonies formed, they were fixed using a 4% paraformaldehyde solution and stained with a 0.1% crystal violet solution. Quantitative analysis was performed using Image J software.
2.7 Single Cell Gel Electrophoresis (Comet Assay)
MGC-803 and HGC-27 cells were embedded in a thin layer of agarose on a microscope slide, creating a "cell-agarose matrix." The slides with the embedded cells were then subjected to RIPA Lysis Buffer (Beyotime, China), which disrupts the cellular membranes and removes cellular proteins, leaving the naked DNA intact. The slides were placed in an electrophoresis buffer and subjected to an electric field. The negatively charged DNA fragments migrate away from the cell nucleus towards the anode. After electrophoresis, the slides were stained with 25 µg/mL of ethidium bromide to visualize the comet-like structures under a confocal laser scanning microscope (Nikon, Japan).
2.8 Mitochondrial Membrane Potential Measurement
Mitochondrial membrane potential assay kit with JC-1 (Meilunbio, China) was used. JC-1 emits red fluorescence in high mitochondrial membrane potential (forming J-aggregates) and green fluorescence in low mitochondrial membrane potential (as monomers). This enables easy detection of mitochondrial potential changes based on fluorescence color transition. MGC-803 and HGC-27 cells in the logarithmic growth phase were collected. The JC-1 dye working solution was added. After a 30-minute incubation in a CO2 incubator, the cells were rinsed 2–3 times with JC-1 dye buffer. The mitochondrial membrane potential was then assessed using a confocal laser scanning microscope (Nikon, Japan).
2.9 Western Blotting Analysis
MGC-803 and HGC-27 cell lines were treated with MLN4924 for 48 h. The cells were then lysed, and the proteins were extracted using RIPA Lysis Buffer (Beyotime, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins based on their molecular weight. The separated proteins were transferred to a nitrocellulose membrane, blocked with milk, and incubated with a specific primary antibody overnight for specific binding to the target protein. After washing, a secondary antibody was added, and the protein signal was detected using an electrochemical luminescence (ECL) detection kit (Meilunbio, China). Signal intensity was quantified using ImageJ software.
2.10 Immunofluorescence
Cells in the logarithmic growth phase were seeded in confocal dishes at a density of 8×104 MGC-803 cells or 5×105 HGC-27 cells per dish. After treatment with MLN4924, 1 mL of 4% paraformaldehyde was added to each dish for fixation, followed by permeabilization with 0.2% Triton, blocking with 5% BSA, and incubation with primary and secondary antibodies. Subsequently, 100 µL of DAPI staining solution (Hoechst 33342) was added to each dish, and the cells were stained at room temperature in the dark for 20 minutes. Finally, the cells were imaged using a confocal microscope. (Nikon, Japan)
2.11 RNA Isolation and Real-time Quantitative PCR
RNA was isolated from cells using Trizol (Solarbio, China) following the manufacturer’s instructions. RNA was treated to remove all genomic DNA, and reverse-transcribed into cDNA using an HiScript II qtr. Kit (Vazyme, China). The cDNA was analyzed using real-time qPCR (SYBR Green; Thermo Fisher, China) with an Applied Biosystems 7900 Sequence Detection System. Each reaction was performed in triplicate. The expression of each gene was normalized by the expression of mouse ribosomal protein β-actin.
2.12 Glucose Uptake-Glo Assay
2-NBDG is a fluorescently labeled deoxy glucose analog used to characterize glucose uptake in cells. Cells in the logarithmic growth phase were seeded in a 6-well plate at a density of 1.5×105 MGC-803 cells or 1.2×105 HGC-27 cells per well. Subsequently, each well was treated with 1 mL of 100 µM 2-NBDG solution and incubated in a light-protected, 37°C cell culture incubator for 20 minutes. After re-suspending in PBS solution, the samples were analyzed using a flow cytometer.
2.13 Transfection of cells with constructs and siRNA GLUT1
MGC-803 and HGC-27 cells in the logarithmic growth phase were seeded into a 6-well plate and transfected using Lipofectamine 2000 Transfection Reagent (ThermoFisher, China) following the manufacturer's protocol. After 6 h of transfection, the transfection mixture was replaced with fresh complete medium. GLUT1 siRNA and HIF-1α siRNA from Jima Co Ltd. (China, China) were used, with the targeted sequences as follows: siGLUT1 5′-CUGCUGAGCAUCAUCUUCATT-3′, 5′-UGAAGAUGAUGCUCAGCACTT-3′; siHIF-1α 5′-CAGGCCACAUUCACGUAUATT-3′, 5′-UAUACGUGAAUGUGGCCUGTT-3′. Scrambled sequences served as the control.
2.14 Metabolite Extraction and Metabolomic Profiling
MGC-803 cells in logarithmic growth phase were seeded and divided into control and 1µM MLN4924 treatment groups. Cells were collected after 48 h and rapidly frozen in liquid nitrogen and stored at -80°C. Subsequently, samples were mixed with extraction solution (Methanol: Water = 3:1 (V/V), containing a mixture of isotopically labeled internal standards), frozen, thawed, vortexed, and sonicated. After centrifugation, the supernatant was collected for analysis. Vanquish UHPLC (ThermoFisher, China)with Waters ACQUITY UPLC HSS T3 column was used for separation. Orbitrap Exploris 120 MS (ThermoFisher, China) performed first and second-level mass spectrometry. Data was converted to mzXML format using Proteo Wizard software. Peak recognition, extraction, alignment, and integration were performed with a self-developed R package (kernel: XCMS). Substances were annotated by matching with the self-established second-level mass spectrometry database BiotreeDB (V2.1), with a cutoff value of 0.3 for the algorithm score.
2.15 In Vivo Assessment of Anti-Tumor Efficacy
Six-week-old female nude mice were procured from SPF Biotechnology (Beijing, China) and provided a standard diet and water ad libitum. All animal procedures strictly adhered to protocols approved by Zhengzhou University's Regulations for Animal Experimentation Platform Management. All experimental protocols were approved by the Animal Ethics Committee of School of Pharmacy, Zhengzhou University. MGC-803 cells were suspended in a 1:1 ratio within DMEM medium supplemented with Matrigel (BD Matrigel, 356234). Subsequently, 4 × 107 cells were injected into the right forelegs of the mice. The mice were randomized into four treatment groups (8 mice per group). They were treated with vehicle, MLN4924 (25 mg/kg), BAY876 (5 mg/kg), or the combined of MLN4924 and BAY876 by intraperitoneal injection or intragastrical administration once a day. Both body weight and tumor volume (mm3) were measured. After a 2-week treatment period, they were euthanized. The serum samples were collected for the purpose of assessing hepatic and renal injuries. Tumors were delicately excised, and samples from the heart, liver, spleen, lungs, and kidneys were procured. These samples were then treated with a 4% PFA Fix Solution (Servicebio, China) to be fixed or flash frozen at -20°C. Hematoxylin and eosin (H&E) staining was performed.
2.16 Statistical Analysis
All experiments were performed with three independent replicates. The obtained data were subjected to statistical analysis using GraphPad, and a two-tailed t-test was employed. Statistical significance was considered for P-values less than 0.05. The levels of significance were denoted as follows: *P < 0.05, **P < 0.01, or ***P < 0.001.