2.1 Reagents
Ziprasidone (Code: MB1289, China) was purchased from MCE (Cat # HY-14542). Antibodies of GOT1, caspase 9, caspase 3, Phospho-JNK-1/2, Phospho-p38, LC3-ii, β-actin, PARP, Cleaved PARP, AKT, PTEN, Erk-1, p-Erk, Bcl-2, Bax, and Bcl-XL were obtained from ABclonal.
Other reagents were as follows: EdU (Guangzhou RiboBio Co., Ltd.), DAPI (Molecular Probes). Cell lines, HepG2 (HB-8065), HCT116 (CCL-247), HCC1806 (CRL-2335), L929 (CRL-6364), PANC-1 (CRL-1469), SW620 (CCL-227), AspC-1 (CRL-1682), SW480 (CCL-228), SW1990 (CRL-2172), A549 (CCL-185), HT1080 (CCL-121) were purchased from ATCC.
2.2 GOT1 protein expression and purification
Briefly, GOT1 protein was expressed by cloning human GOT1 ORF (GeneBank: NC_000010.11) into a pET28a vector containing 6 His-tag. The recombinant plasmids were transduced into E. coli strain BL21 and cultured in Luria-Bertani medium at 37 °C, and then 0.4 mM IPTG was added at 18 °C to induce GOT1 protein expression for 18 h. The details of GOT1 protein purification were as previously reported [14, 15]. The purified GOT1 protein is quick-frozen and stored at -80°C for subsequent experiments.
2.3 GOT1 inhibition assay
An enzyme activity reaction system was constructed in vitro to evaluate the inhibitory activity of ziprasidone on recombinant GOT1 protein by monitoring the change in absorbance at 340 nm caused by the reduce of NADH on Microplate Reader (BioTek, USA). Total 100 μL reaction system, contained 0.1 mg/mL GOT1, 1 mM α-KG, 4 mM aspartic acid, 1 mM NADH and 1 unit/mL malate dehydrogenase [15].
2.4 Microscale Thermophoresis (MST) assay
The binding affinity between ziprasidone and GOT1 were determined by MST according to the instruction of Monolith™ NT.115 Protein Labeling Kit RED-NHS (Cat # MO-L011). GOT1 was dispersed into 20 mM HEPES (pH 7.5) solution with final concentration of 10 μM and labelled by the RED-NHS dye. Ziprasidone was diluted to different concentrations based on gradient dilution and was incubated with the labeled GOT1 for 20 min. All samples were tested on the Monolith NT.115 instrument for their binding affinity to GOT1 (Nano Temper Technologies, Germany) [15].
2.5 Molecular Docking
The crystal structure of GOT1 (PDB ID: 3II0) was downloaded from the Protein Data Bank (http://www.rcsb.org/). The docking was executed through ICM 3.8.2 modeling software (MolSoft LLC, San Diego, CA). The ligand binding sites was selected though ICM software graphical tools for the molecular docking. The potential energy diagram of the receptor was calculated with default parameters. According to the Monte Carlo procedure 30, the numerous conformations of ligands were tested. Finally, the most promising conformations of the ligand were selected with the lowest-energy [16].
2.6 Cell viability
The MTT assay was used to evaluate the cell viability after compound treatment. 5 × 103 cells/well were seeded in 96-well plates until it converges to 80%, and then administrated with different concentrations of ziprasidone (0-100 μM). 100 μL of 5 mg/mL MTT was added after ziprasidone treatment for 24 h. After incubated for 4 h at 37oC, DMSO (100 μL/well) was added to dissolve the formazan crystals. The absorbance was measured at 490 nm with a microplate reader.
2.7 Apoptosis analysis
After treating SW1990 cells with 20 μM and 40 μM ziprasidone for 24 h, the cells were collected and incubated with Annexin V-FITC/PI for staining. The specific operation details referred to the Roche Annexin-V-FLUOS Staining Kit (Cat # 11858777001). Fluorescence was determined by FACS Verse flow cytometer.
2.8 Colony formation assay
SW1990 cells were inoculated in a 6-well plate at 200/well and cultured for 24 h. Then, SW1990 cells were treated with different concentrations of ziprasidone. During the culture, the fresh medium containing ziprasidone was changed every three days until visible colonies were formed. SW1990 colonies were stained with 1% crystal violet, then washing with PBS for three times and counting colonies containing more than 50 cells.
2.9 Immunofluorescence staining
Briefly, cells were seeded into 96-well plates for 12 h and were treated with different concentrations of ziprasidone for 24 h, 4% paraformaldehyde for 20 min, 0.2% Triton X-100 for 10 min, and incubated with 50 μM EdU for 2 h. The EdU proliferation experiment was performed according to the kit instruction of the previous description [17].
For the Hoechst 33342 staining, SW1990 cells were seeded into 96-well plates with 1 × 104/well and cultured for 12 h, then replaced with fresh DMEM containing DMSO or 10-40 μM ziprasidone, cultured for another 12 h, and dyed with 100 μL 20 μM Hoechst 33342 for 10 min. The morphology of nuclear chromosomes was observed under a fluorescence microscope at 340 nm. (Nikon, Japan).
2.10 Transwell migration
First, 600 μL complete medium with ziprasidone were supplemented into the lower chamber, and then 5 × 104 SW1990 cells were dispersed in 100 μL DMEM medium without FBS in upper chamber. The cell colonies at the bottom of the membrane were stained with crystal violet and then observed and counted under a microscope (Nikon, Japan).
2.11 Wound scratch assay
SW1990 cells were cultured until the cell density reached about 85%, three straight scratches were made by 10 μL micro pipette tip per well. Wash the 6-well plates with PBS before treating the cells with different concentrations of ziprasidone. At 0 h and 24 h after ziprasidone treatment, microscopic pictures were taken to analyze the compound's ability to inhibit cell migration.
2.12 Western blot assays
SW1990 cells were seeded into a 6-well plate at 5 × 105/well and cultured until the cell confluence was above 85%. SW1990 cells were treated with ziprasidone for 24 h and then collected. Cells were resuspended with RIPA lysis buffer (Beyotime Biotechnology) containing 0.1% PMSF to release total protein. The concentration of total protein was determined by BCA Protein Assay Kit (Beyotime Biotechnology). Total protein was separated by SDS-PAGE and transferred to PVDF membrane (Merck Millipore, 0.2 μm). The PVDF membrane was incubated with the primary antibody overnight at 4°C, and then with the secondary antibody for 2 h at room temperature, finally performed chemiluminescence imaging (Tanon 5200).
2.13 siRNA knockdown of GOT1
SW1990 cells were seeded into a 6-well plate at 5 × 105/well and cultured until the cell confluence was 90%-95%. Mix 100 pM siRNA with 5 uL Hieff Trans™ Liposomal Transfection Reagent and incubate for 20 min at room temperature. siRNA-liposome complexes treated SW1990 cells for 6 h and then replaced them with fresh DMEM medium and continued to culture for 48 h.
siRNA-1 Forward: 5'to3' CAG GUA AUG UGA AGA CAA UTT
siRNA-1 Reverse: 5'to3' AUU GUC UUC ACA UUA CCU GTT
siRNA-2 Forward: 5'to3' GCG CGU UGG UAC AAU GGA ATT
siRNA-2 Reverse: 5'to3' UUC CAU UGU ACC AAC GCG CTT
siRNA-3 Forward: 5'to3' AGA AAG UGG AGC AGA AGA UTT
siRNA-3 Reverse: 5'to3' AUC UUC UGC UCC ACU UUC UTT
2.14 Cellular thermal shift assay (CETSA)
CETSA (cellular thermal shift assay) is usually used to evaluate the targeting effect between the compound and the protein based on the principle that the ligand can improve the thermal stability of the target protein. SW1990 cells were cultured to more than 80% confluence, replaced with fresh DMEM containing DMSO or 30 μM ziprasidone, and cultured for another 12 h. SW1990 cells were heat-treated from 42°C to 52°C for 3 min. Specific experimental details referred to the previous description [18-20].
2.15 Measurement of ECAR and OCR
SW1990 cells were seeded in 24-well plates at a density of 5 × 104 and cultured for 12 h, and then treated with fresh DMEM medium containing 20 μM ziprasidone for 3 h. XF24 Extracellular Flux Analyzer (SeaHorse Bioscience) was used to determine the changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of SW1990 cells after administration of ziprasidone. Experimental details were as previously reported [12].
2.16 Metabonomics experimental methods
The SW1990 cells were confluent to more than 80% and treated with 20 µM ziprasidone, and the control group was added with an equal volume of DMSO. After culturing for 24 h, we collected the cells and added 70% methanol aqueous solution to lyse on ice. The cells were lysed by repeated freezing and thawing of liquid nitrogen and the metabolites were dissolved in methanol aqueous solution. The supernatant is centrifuged to perform LC-MS/MS analysis. Parameter settings and operating mode for LC-ESI-MS/MS analysis were as previously described [17].
2.17 In vivo tumor xenograft study
CB-17/SCID mice (male, 4 weeks old) were s.c. inoculated with 3 × 106 SW1990 cells in the left abdomen. The tumors were allowed to grow for 6 d. The mice inoculated with tumor cells were randomly divided into three groups with 10 mice/group, including the control group, the low-dose ziprasidone group (100 mg/kg), and the high-dose ziprasidone group (200 mg/kg). Mice were given intragastric administration once a day. Tumor volume measurement and mouse body weight measurement were done as previously described [12].