Sorafenib was kindly provided by Bayer Pharmaceuticals. YC-1 was obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). Anti-PARP, anti-caspase 8, anti-β-actin, and anti-caspase 9 were obtained from Beyotime (Jiangsu, China). Horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit secondary antibodies were from Santa Cruz (Dallas, TX, USA). All other antibodies were purchased from Abcam (Cambridge, TX, USA).
Cell lines and cell culture
Established human HCC cell lines, HepG2 and BEL-7402 were from the American Type Culture Collection (ATCC; Manassas, VA, USA). HCCLM3 was kindly provided by the Liver Cancer Institute and Zhongshan Hospital (Shanghai, China) . All HCC cells were maintained in high-glucose Dulbecco’s modified Eagle medium (DMEM) supplement with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies, Cergy Pontoise, France) in a humidified atmosphere of 5% CO2 at 37°C. The human normal liver cell line L02 was provided by Cancer Institute & Hospital of Chinese Academy of Medical Sciences, which was cultured in PRMI 1640 supplement with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. All cell lines were immediately expanded and frozen down such that all cell lines could be restarted every 3 months from a frozen vial of the same batch of cells. No further authentication was done. All cell lines were routinely tested to rule out mycoplasma infection.
Cell proliferation assay was measured using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Shanghai, China) according to the manufacturer’s instructions. Briefly, cells were cultured in 96-well plates at a concentration of 3 × 103/well, incubated for 24 h, and treated with sorafenib and/or YC-1. After 72 h treatment, CCK-8 reagent was added to each well. The absorbance was measured at 450 nm after 2.5 h incubation at 37°C using an automated ELISA plate reader. Any synergistic effects resulting from combination of the compounds were measured using the methods described by Chou and Talalay by using Microsoft Excel software to determine the combination index values (CI > 1: antagonistic effect, CI = 1: additive effect, and CT < 1: synergistic effect) .
Colony formation assay
Briefly, 6-well dishes were seeded with 1 × 103 viable cells and allowed to grow for 24 h. The cells were then incubated in the presence or absence of sorafenib, YC-1 and their combinations for 24 h in complete medium, washed with media, and allowed to grow in complete medium for 2 weeks. The colonies obtained were washed with PBS and fixed in 4% paraformaldehyde for 20 min at room temperature and then washed with PBS followed by staining with crystal violet. The colonies were counted and compared with untreated cells. Three different independent experiments were performed.
Cell cycle analysis
HCC cells were plated in 6-well plates at 2 × 105 cells/well. Following the designated treatments, cells were harvested by trypsinization and washed with PBS and fixed in ice-cold 75% ethanol overnight at -20°C. Fixed cells were washed, and dissolved in RNAse and subsequently incubated at 37°C for 30 min. Next, cells were stained with propidium iodide (PI) for 30 min. The DNA content of the cells (1 × 104 cells per experimental group) was determined using a BD Accuri C6 flow cytometer (BD biosciences).
HCC cells (HepG2, BEL-7402 or HCCLM3) were seeded in 6-well culture plates in culture medium with 2% FBS at the concentration of 2 × 105 cells/well. The following day, the cells were treated with sorafenib and/or YC-1 for a 48 h period. After treatment, cells were washed with PBS and fixed with 4% paraformaldehyde followed by staining with Hoechst 33258, and the apoptosis cells were examined by immunofluorescence microscope, or all cells including both floating and attached cell were collected, and the apoptotic cells were detected by Annexin V-FITC Apoptosis Detection Kit (KeyGEN Biotech, Nanjing, China). The cells were stained with Annexin V-FITC and PI according to the supplier’s instructions. Viable and dead cells were detected by a BD Accuri C6 flow cytometer (BD biosciences).
Gene knockdown using small interfering RNA
SMARTpool small interfering RNAs (siRNA), including control and STAT3 were synthesized by Shanghai GenePharma Co. (Shanghai, China). The procedure has been described previously .
Western blot analysis
Cell lysate protein content was determined using a Bicinchoninic acid (BCA) protein assay kit. Equivalent amounts of whole cell extracts, which were based on total protein content, were subjected to SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 2 h and then incubated with respective primary antibody overnight at 4°C followed by the incubation with the appropriate HRP-conjugated secondary antibody for 1.5 h at room temperature. Immunoreactivity was detected with SuperSignal West Pico substrate (Thermo scientific, Rockford, IL, USA).
SHP-1 phosphatase activity
The Rediplate 96 EnzChek®Tyrosine Phosphatase Assay Kit (R-22067) was used for SHP-1 activity assay (Molecular Probes, Carlsbad, CA). The SHP-1 phosphatase activity was measured as described before .
Male BALB/c nu/nu mice (4–6 weeks of age) were obtained from Vital River Laboratories (Beijing, China) and housed under defined flora conditions in individually ventilated sterile microisolator cages. All experimental procedures using these mice were carried out in accordance with protocols approved by the Animal Care and Use Committee of Capital Medical University (Beijing, China).
Orthotopic and ectopic HCC models
In the orthotopic model, HCCLM3 cells (5 × 106) were suspended in 200 μL serum-free DMEM and matrigel (1:1) and then injected subcutaneously into the upper right flank region of nude mice. When the subcutaneous tumor reached approximately 1 cm in length (approximately 4 weeks after injection), it was removed, minced into small pieces of equal volume (2 × 2 × 2 mm3), and transplanted into the livers of 20 nude mice. In the ectopic HCC model, HepG2 cells (5 × 106) were suspended in 200 μL serum-free DMEM and matrigel (1:1) and then injected subcutaneously into the upper right flank region of 20 nude mice. When the tumor reached a mean size of about 100 mm3, mice were randomized into each experimental group according to tumor size, to start the treatment with a similar mean size in each group. Mice were treated with sorafenib by oral route (30 mg/kg/day), or intraperitoneal YC-1 (10 mg/kg/day), or combination of sorafenib and YC-1, or DMSO and polyoxyethylenated castor oil as control every day for up to the 24th day. Tumor size was measured with a caliper rule every 3 days. The tumor volume was calculated as follows: TV (mm3) = (L × W2)/2, where L was the longest and W the shortest radius of the tumor in millimeters. At the end of the experiments, mice were euthanized, blood samples were collected via cardiac puncture, and tumor tissues were removed for fixation in the 4% paraformaldehyde for histologic examination and immunohistochemical staining.
Tissues were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. Paraffin-embedded tissue sections were cut into standard 6 μm sections, deparaffinized in xylene and rehydrated through graded alcohol solutions. Antigen retrieval was performed 10 min at 92°C in EDTA (10 mmol/l, pH 8.0) in a water bath. Endogenous peroxidases were inactivated by immersing the sections in 0.3% hydrogen peroxide for 12 min. The sections were blocked with 5% goat serum for 60 min at 37°C. The slides were incubated with primary antibodies for overnight at 4°C. Next, the slides were treated with appropriate HRP-conjugated secondary antibody for 40 min at 37°C and then developed with 3,3′-diaminobenzidine. Finally, the slides were counterstained with hematoxylin and mounted. The slides were examined with Nikon Eclipse Ti microscope under a 200× objective.
All values are expressed as the mean ± SEM. The data were analyzed using Student’s t test or the ANOVA test. A P value of <0.05 was considered statistically significant. GraphPad Prism (GraphPad Software Inc., San Diego, California, USA) was used for these analyses.