Cell culture and reagents
The Human prostate cancer cells lines PC3 and DU145, purchased from the Cell bank of the Chinese Academy of Sciences, were grown in RPMI-1640 medium and minimum essential medium (MEM). For cell culture, medium was supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (MRC) and maintained at 37 °C in an atmosphere of 5% CO2 in air. L-ascorbic acid (Alfa aesar) was diluted in phosphate buffered saline (PBS) and neutralized with sodium hydroxide before use. ASNase was also dissolved in PBS, frozen in -20℃ refrigerator, and thawed when used .
WST-8 cell viability assay
The WST-8 assay was used to quantify cell viability following incubation of cells with ASNase/vitamin C. WST-8 tetrazolium salt was reduced by cell dehydrogenase to methoxypyrimidine (Orange). By measuring its absorbance (450 nm), formazan is then quantified. It indicates that the number of metabolically active (alive) cells. According to the manufacturer's instructions (DOJINDO Laboratories, Kumamoto, Japan), the WST-8 assay was carried out to monitor cell proliferation.PC3 or DU145 cells were seeded in 96-well plates with the density of 104 cells and then incubated with different drugs for the indicated time periods. Then, add 100ul WST-8 into the medium in each well and incubate the well for 1-2 hours. Finally, Absorbance was measured at 450 nm using a Multiskan FC apparatus (Thermo Fisher Scientific, Waltham, MA, USA) .
Cells (2-3×105 cells/well in 3ml) were first incubated with or without ASNase (1U/ml) for 24h at 37°C. Then, vitamin C was administered at the following concentrations:0,1,2,4,8 mM. After 16h, the cells were harvested with 0.05% trypsin solution, washed twice with PBS and centrifuged at 1,500 rpm for 5 min. Next, the cells were stained with fluorescein isothiocyanate-labeled Annexin V (BD Pharmingen, San Jose, CA, USA) and counterstained with propidium iodide (PI, BD Pharmingen), resuspended in binding solution. Finally, the cells were analyzed using flow cytometry (CytoFLEX S, Beckman Coulter, Fullerton, CA, USA), as referenced to Li et al .
Western blot analysis
DU145 and PC3 cells were washed, collected and lysed. Separate the proteins with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transfer onto a polyvinylidene fluoride (PVDF) membrane. First, block the sample with 5% nonfat milk powder for one hour. Rabbit anti-glutamine synthetase antibody (Abcam) were diluted to 1:10000 in blocking buffer and incubated with the membranes overnight at 4°C and use mouse anti-β-tubulin as internal reference protein. Then, wash the membranes and incubate the membranes with a secondary antibody. After incubation, ELISA determination as described previously .
The glutamine synthetase (GS) ELISA kit was purchased from Wuhan USCN Life Science Company. The culture medium containing cells was centrifuged at 1000 rpm for 5 min, and the supernatant was taken out, and 100% was added to each well with 100μL test solution A, warm bath at 37 ℃ for 1 h, then discard the liquid in the hole, and use 350μL washing liquid for each hole. Add 100 μL solution B to each hole, and finally 90 μL TMB substrate solution and 50 μL termination solution. The optical density of each well was measured at 450 nm by microplate immediately.
NADP/NADPH Assay Kit was purchased from Abbkine Company. Cells treated with 2mM VC were collected after trypsinization. Then, the intracellular NADP+ and NADPH of PC3 and DU145 were extracted and placed on ice, adding prepared 80ul working fluid quickly. Tap plate to mix briefly and thoroughly. Read optical density at 565nm using Multiskan FC apparatus (Thermo Fisher Scientific), and after a 30-min incubation at room temperature, the NADPH/NADP+ ratio was calculated as instructions described (Thermo Fisher Scientific) .
Xenograft mouse model
BALB/c nude mice (5-week-old male) were purchased from the Model Animal Research Center of Nanjing University. The animal experiments were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Sun Yat-sen University and conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. BALB/c male nude mice were randomly divided into four groups. Each mouse was injected subcutaneously with 5*106 PC3 or DU145 cells on both sides. Once the tumor reached a size of ~50 mm3, the mice were respectively administered with vehicle, VC (Sodium ascorbate), ASNase, or the combination of VC and ASNase; Sodium ascorbate was purchased from Sigma Aldrich (St. Louis, MO, USA). ASNase (dose=60U/ml) was administered intraperitoneally daily in all experiments. VC (dose = 4 g/kg) was administered intraperitoneally twice a day in all experiments. Tumor sizes (length×width2×0.5) were measured once every 2 days. After 16 days the tumors were excised, weighed and analyzed by immunohistochemistry. No adverse effects were observed in the animals .
13N-ammonia (Synthesis see above) was administered via tail vein (100 μ L) at an active dose of 1 mCi per mouse one day and three days after treatment initiation. After the injection, computed tomography (CT) scanning was started immediately, imaging was conducted using a micro-PET system (Inveon, SIEMENS, Germany). The mice were then imaged for a 15-min static acquisition. Regions of interest were drawn around tumors (Ts) and the contralateral normal tissues (NTs), and the tumor-to-background ratios (T/NT) were calculated.18F-FDG was administered via tail vein injection (100 μL) at an activity dose of 100 µCi per mouse one day before,one week and two weeks after treatment initiation. Different from 13N-ammonia, 18F-FDG was allowed to accumulate in the tumor for 45 min. PET-CT scans were performed as shown above .
Tumor tissues were collected for IHC at the end of treatment. Tumor tissues were cut to 4-mm thick slices and incubated with 3% hydrogen peroxide for 25 min at room temperature to block endogenous peroxidase activity. Next, the tissues were incubated with bovine serum albumin (BSA) for 30 min at room temperature to block nonspecific binding. Afterward, sections were incubated overnight at 4°C with primary antibody (purified mouse anti-GS, 1:1000), then incubated with biotinylated secondary antibody (HRP-labeled goat anti-rabbit IgG, 1:200) followed by streptavidin biotin peroxidase complex (streptavidin biotin peroxidase complex immunohistochemical kit) for 30 minutes at room temperature. The immunoreactions were visualized using a Dolichos Biflorus Agglutinin (DBA) chromogenic reagent kit after incubation for 10 min at room temperature, followed by hematoxylin staining for 3 min as reported by Long et al .
Comparisons between two groups were carried out using Student’s t test or analysis of variance (ANOVA) with Bonferroni post hoc test. Each experiment was repeated at least in triplicate, and mean ±SEM was calculated for each value. For all analyses, p < 0.05 *, < 0.01 ** or < 0.001 *** between exposure conditions were considered significant. All analyses were performed in GraphPad Prism (GraphPad Software, Inc.).