GLS1 expression in endometrial cancer
Publicly available data were retrieved and analyzed in the context of this study. Transcriptomics (microarray) data from 877 cancer cell lines retrieved from the Cancer Cell Line Encyclopedia (CCLE) [http://www.broadinstitute.org/ccle]. RNA sequencing data from uterine corpus endometrial carcinoma (UCEC) patients were downloaded from The Cancer Genome Atlas (TCGA) database using Broad GDAC Firehose [https://gdac.broadinstitute.org]. GLS1 expression levels in different stages of endometrial cancer progression and normal endometrium were assessed using Gene Expression Profiling Interactive Analysis (GEPIA) [http://gepia.cancer-pku.cn/index.html].
Cell culture and reagents
The human endometrial cancer cell lines Ishikawa and HEC-1B were purchased from the China Center for Type Culture Collection (Wuhan, China). The Ishikawa cell line was maintained in MEM medium (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS) (Gibco, Invitrogen). HEC-1B cells were maintained in RPMI 1640 medium (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 5% FBS. All cell culture media were supplemented with 100 U/ml of penicillin and 100 μg/ml of streptomycin. Cells were cultured in a humidified 5% CO2 atmosphere at 37°C.
Compound 968 was purchased from Calbiochem (Billerica, MA) and diluted with DMSO to make up stock solutions of 10 mmol/L. The anti-GLS1 antibody was purchased from Abcam (1:1000), and all the other antibodies were obtained from Cell Signaling Technology (1:1000).
Colony formation assay
Ishikawa and HEC-1B human endometrial cancer cells were harvested at a log phase of growth. Cells were seeded into 6-well plates (800 cells/well) and were incubated for 24 h in their standard growth media to allow for cell adhesion. After incubation, the cells were treated with compound 968 (0, 5, 10, and 25 μM) for 24 h, followed by the replacement of the media with drug-free cell culture media. Cells were further incubated at 37°C for 14 days, during which the cell culture media was refreshed every three or four days. After incubation, cells were subjected to 0.5% crystal violet staining, and colony quantification was achieved by counting under a light microscope. The treatments were performed in triplicates, and the clonogenic assays were repeated three times.
Cell proliferation assay
The cell viability of human endometrial cancer cells was assessed in MTT assays, as previously described[14]. Briefly, Ishikawa and HEC-1B cells harvested at the logarithmic growth phase were seeded into 96-well plates (4000 cells/well) in regular media and allowed to adhere for 5 h. After the incubation, cells were treated with increasing concentrations of compound 968, ranging between 2 and 50 μM for 48 h. Alternatively, cells were incubated in the presence of 2 μM or 10 μM of compound 968 for 24, 48, 72, 96, and 120 h. The cells were treated with different concentrations of paclitaxel (0, 0.1, 1.0, 10, 50 nM) in media with 10 μM compound 968 for 72 h. To quantify cell viability, MTT was added in each of the wells of the 96-well plate at a final concentration of 0.25 mg/ml. After one hour of incubation, the MTT-containing cell culture media was replaced by 100 μl of DMSO, and the optical absorbance at 570 nm was measured on a plate reader. The treatments were performed in triplicates, and the experimental procedure was repeated three times.
Annexin-V assay
The ability of compound 968 to induce cell apoptosis was examined by flow cytometry using the Annexin-V FITC kit (BD Biosciences Pharmingen, San Diego, USA) and following the instructions of the manufacturer. In brief, human endometrial cancer cells were seeded into 6-well plates at 2 × 105 cells/well and allowed to adhere for 5 h. Cells were then exposed to increasing concentrations of compound 968 (0, 5, 10, 25 μM). After a 24-hour incubation, the cells were harvested with EDTA-free 0.25% trypsin and washed with PBS. Subsequently, cells were incubated in 100 μl of Annexin-V/PI solution for 15 min in the dark and analyzed on a FACS Calibur instrument. The flow cytometry results were analyzed using the FlowJo Software 7.6 (TreeStar). At least two experimental repeats were conducted.
Cell cycle analysis
As described above, 2 × 105 cells/well were seeded into 6-well plates for 5 h, and then the cells were treated with increasing concentrations of compound 968 for 48 h. After incubation, the cells were harvested using 0.05% trypsin, washed with phosphate-buffered saline (PBS) solution, and fixed using 90% methanol; fixed cells were kept at -20°C until they were subjected to cell cycle analysis by flow cytometry. Before flow cytometry, residual methanol was removed by washing with PBS. After the cells were centrifuged, they were incubated in 50 μl of RNase A solution (250 μg/ml) containing 10 mM EDTA for 30 min at 37°C. Subsequently, cells were resuspended in 50 μl of propidium iodide (PI) staining solution, which contained 2 mg/ml PI, 0.1 mg/ml Azide, and 0.05% Triton X-100, and incubated for 10 min protected from light. The stained cell samples were analyzed on a FACS Calibur instrument (Becton Dickinson). The flow cytometry results were analyzed using the FlowJo Software, version 7.6. At least two independent repetitions were performed for each condition.
Reactive oxygen species (ROS) generation analysis
The ability of the compound 968 to affect ROS generation and induce cellular oxidative stress in human endometrial cancer cells was assessed by measuring the intracellular ROS levels using the ROS-sensitive fluorescence indicator DCFH-DA (Jiangsu KeyGEN BioTECH Corp. Ltd, Jiangsu, China) and flow cytometry, as described previously (Eruslanov E and Kusmartev S, 2010). In brief, Ishikawa and HEC-1B cells were seeded in 6-well plates at 2.5 × 105 cells/well. After cells were allowed to adhere for 5 h, the cells were treated with 5, 10, and 25 μM of compound 968 for 12 h. Subsequently, cells were incubated in the presence of DCFH-DA (20 μM) for 20 min, and then harvested, washed with PBS, and then analyzed on the FACS Calibur instrument. The flow cytometry results were analyzed using the FlowJo Software, version 7.6. At least two independent repetitions were performed for each condition.
Invasion assay
The effects of compound 968 treatment on the invasion capacity of human endometrial cancer cells were assessed using transwell chambers with pores of 8 μm in dimeter (Costar, NewYork, NY, USA), as standard methodologies described elsewhere[16]. Briefly, serum-starved (for 12 h) Ishikawa and HEC-1B cells were seeded into the upper chambers of 24-transwell inserts at 50,000 cells/well, followed by a 12-hour incubation in 50 μl of serum-free medium; during the incubation, the lower chambers were covered with 150 μl of regular cell growth media containing different concentrations of compound 968. To allow for sufficient time for the invasion into the lower chamber to occur, the plates were incubated for 24 h at 37°C. After incubation, a cotton swab was used to remove any cells that did not migrate through the membrane. The cells that had successfully invaded into the lower chamber were fixed and stained using 0.1% crystal violet. Images from five representative fields were acquired using a light microscope, followed by cell quantification. At least two independent repetitions were performed for each condition.
Western blot analysis
Total protein was extracted from endometrial cancer cells using RIPA buffer (Cwbiotech, CW2333S, China), and the protein was quantified with the BCA assay kit (Cwbiotech, CW0014S, China). Equal amounts (20 μg) of the protein were separated in a 12% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% nonfat milk and then incubated overnight at 4°C with 1:1000 dilution of primary antibodies. The membranes were washed and incubated with a secondary peroxidase-conjugated antibody for 1 h at room temperature. The membranes were visualized using ECL blotting detection reagents (Bio-Rad, USA). Relative protein contents were quantified using β-actin as a loading control. Each experiment was repeated at least twice.
Tumor xenograft growth assay
Four-week-old BALB/c nude female mice (Beijing HFK Bioscience Co., Ltd., Beijing, China) with severe combined immunodeficiency were used for our experiments. A total of 1x107 Ishikawa cells in 0.1 ml of serum-free MEM were injected subcutaneously into the right flank of the mice. Two weeks after injection, the mice were treated with intraperitoneal injections of compound 968 (5 mg/kg) every day for two weeks. Subcutaneous tumors were dissected and weighed. Tumor volume was calculated using the following equation: tumor volume = (width2 ×length)/2. All mice were handled according to protocols approved by the Institutional Animal Care and Use Committee of Shandong Cancer Hospital and Institute, China. All methods were performed in accordance with the NIH guidelines and regulations.
Immunohistochemical analysis and scoring
Mouse tumor tissues were formalin-fixed and paraffin-embedded. Slides (4 μm) were treated with xylene and rehydrated, and then 3% hydrogen peroxide was used to eliminate endogenous peroxidase activity. Antigen retrieval was performed through boiling in a microwave, and 5% bovine serum albumin was applied to block nonspecific binding. Slides were incubated with the primary antibodies of rabbit anti-GLS1 (1:1000, Abcam), rabbit anti-phosphor-S6 Ribosomal protein (Ser235/236) (1:1000, Cell Signaling Technology), rabbit anti-cyclinD1 protein (1:1000, Cell Signaling Technology) and rabbit anti-E-cadherin (1:1000, Cell Signaling Technology) at 4°C overnight. At least 3 pieces 200× views were randomly selected for taking pictures each slide in each group. Try to make the tissue fill the whole field of view when taking pictures, make sure that the background light of each photo is same. Use Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) to select the same brown-yellow color as the uniform standard for judging the positive of all photos, and analyze each photo to get the integrated optical density value (IOD) and positive pixel area (AREA) of each photo. And calculate the average optical density (average optical density, AOD value), AOD = IOD / AREA, the larger the AOD value, the higher the positive expression level.
Statistical analysis
Uterine Corpus Endometrial Carcinoma (TCGA, PanCancer Atlas; n = 527) RNA sequencing and clinical data were retrieved using Broad GDAC Firehose [https://gdac.broadinstitute.org]. Overall survival and progression-free survival analyses were performed using the Kaplan-Meier method. Statistical significance in differences in survival was determined by the log-rank (Mantel-Cox) test using GraphPad Prism version 7.0c. Data are expressed as mean ± standard error. Comparisons between two groups were performed using the two-tailed Student’s t-test. Significance was set at *P < 0.05, **P < 0.01.