Chemicals and reagents
4SC-202 was provided by Lan-jun Biotechnology Co.Ltd (Shanghai, China). Cell Counting Kit-8 (CCK-8) kit and DMSO were purchased from Sigma Aldrich (MO, USA). All cell culture reagents were purchased from Gibco Life Technologies (Grand Island, CA, USA).
Cell culture
Cervical cancer cell lines SiHa, Hela, and CaSki were obtained from the American Type Culture Collection (Manasas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml). Cell culture was conducted in a humidified atmosphere of 5% CO2 at 37°C. Cells were grown to 80% confluence for further assays. All culture reagents were obtained from Gibco (Carlsbad, CA).
CCK-8 assay
SiHa, Hela, and CaSki cells were plated in 96-well plates and incubated with various concentrations of 4SC-202 (0, 0.01, 0.1, 1, 5, and 10 μM) for a certain period of time (24 h, 48 h, or 72 h). CCK-8 reagent (10 μL) was added and the mixture was incubated for 2 h in the dark. The optical density (OD) value for each well was measured at a wavelength of 450 nm. The half maximal inhibitory concentration (IC50) was calculated by using SPSS software.
Colony formation assay
Colony formation assay was carried out as described previously [18]. SiHa cells (5×103 cells/well) were plated in 6-well plates and incubated with various concentrations of 4SC-202 (0, 1, and 5 μM) for 72 h. Culture media were refreshed every 2 days. The cells were stained with crystal violet, and the colonies were photographed and manually counted on day 10.
Cell cycle analysis and apoptosis detection
For cell cycle analysis, SiHa cells were incubated with various concentrations of 4SC-202 (0, 1, and 5 μM) for 72 h, digested with 0.25% trypsin and resuspended in phosphate-buffered saline (PBS) at a density of 1 × 106 cells/mL. Cell cycle distribution was detected by flow cytometry (BD Biosciences, CA, USA) and analyzed using Cell Quest software. For apoptosis detection, the cell suspension was gently mixed with 5 μL Annexin V-FITC and 10 μL PI, followed by an incubation at room temperature for 15 min in the dark. Apoptosis rate was determined by using the flow cytometry.
Western blotting
Total proteins were extracted from the cell lines using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). Tissue lysates were centrifuged at 14,000 rpm for 5 min at 4°C, and the supernatants were collected for further analysis. The protein concentration was determined using BCA protein Assay Kit (Beyotime). Fifty μg of total protein were mixed with loading buffer, denatured at 95°C for 5 min, resolved on a 10% SDS polyacrylamide gels and electro-transferred to PVDF membranes (Bio-RAD, CA). After being blocked with 5% milk and 1% bovine serum albumin solution, the membrane was incubated at 4°C overnight with the following primary antibodies: anti-Cleaved-caspase3, anti-BCL-2, anti-Bax, anti-p-STAT5a, anti-STAT5a, anti-p-AKT, anti-AKT, anti-p-ERK1/2, anti-ERK1/2, and anti-GAPDH (Acbam, diluted 1:1000). On the following day, the membrane was washed 3 times with TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies (Acbam, diluted 1:5,000) for 2 h at room temperature. The protein bands were developed with a chemiluminescence detection system (Immobilion, Millipore), and the relative band intensity was analyzed using ImageJ software.
Immunofluorescence
Cultured cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Fixed cells were blocked with 1% BSA at room temperature for 30 min. After the pretreatments, the cells were incubated with the primary antibodies (PRLR 1:20) at 4°C overnight, followed by an incubation with Alexa Flour-conjugated secondary antibody (Invitrogen) at room temperature for 1 h in the dark. Nuclear DNA was counterstained with 10 ng/ml DAPI at 4°C for 30 min in the dark. Stained cells were observed and photographed under a fluorescence microscope (200 ×).
Xenograft assay
Five-week-old female C57BL/6 nude mice were obtained from Bioscience (Beijing, China) and housed in a laboratory animal room under standard conditions (12-h light/dark cycle, 20-25°C, and 60-85% humidity), with ad libitum access to sterilized food and water. The mice were randomly divided into three groups (n = 5 per group): control, 50 mg/kg 4SC-202, and 100 mg/kg 4SC-202. SiHa cells were injected into the upper right flank of 15 nude mice. 4SC-202 treatment was started on day 6 post-injection when the tumors were palpable. 4SC-202 was dissolved in DMSO and administered by gavage every two days. The tumor size was measured every 3 days, and tumor volumes were calculated as follows: (V) = width2 × length × 0.5. In the meantime, body weights of the mice were recorded every week. All the animals were euthanized, and the xenografts were weighted after 30 days.
Subcutaneous tumors were dissected, removed and kept for immunohistochemical and western blot analyses. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by AU680 Chemistry System (Beckman Coulter, Brea, CA, USA). Organ tissues including lung, liver, kidney, and heart were removed, sectioned and subjected to H&E staining. Animal experiments were approved by the Ethics Committee of Medical Laboratory Animal Center of Chinese People's Liberation Army General Hospital and performed in accordance with National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals.
Immunohistochemical analysis
Paraffin-embedded sections were dewaxed with dimethyl benzene and rehydrated in dH2O. The slides were immersed in citrate buffer (0.01 M, pH 6.0) with high-pressure heating for 2 min. After being blocked with 1% BSA (Sigma, USA) in TBST buffer for 5 min at room temperature, the slides were incubated with primary antibodies against Ki-67 (Proteintech, Wuhan, China) at a dilution of 1:16000 overnight at 4 °C, followed by an incubation with goat anti-rabbit IgG secondary antibody (Thermo Fisher, 1:5000 dilution) and DAB staining. Finally, the slides were counterstained with hematoxylin, dehydrated and sealed with neutral gum. Organ tissues were stained with H&E for histological analysis. The images were captured under a Nikon microscope.
Statistical analysis
Results were presented as the mean ± standard deviation (SD). At least three experimental replicates were evaluated for each sample. The significance of the experimental data was analyzed by using PRISM® GraphPad 8.0 software, one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc tests. The significance level was set at P<0.05, and p-values were indicated accordingly.