Reagents and antibodies
Sorafenib was obtained from MedChem Express (Monmouth Junction, NJ, USA). ERK1/2 inhibitor (LY3214996) was purchased from Selleckchem (Houston, TX, USA). Antibodies against cyclin D1 and phospho-Rb were purchased from Abcam Biological Technology (USA). Phospho-ERK1/2 Kit (#9911) and antibodies to ERK1/2, Caspase-3, Cleaved Caspase-3, Caspase-9, Cleaved Caspase-9, PARP, Cleaved RARP, PI3K110β, Akt, phospho-Akt (Ser473), mTOR, phospho-mTOR (Ser2481), Bim, Bad, Bak, Bax, P70S6K, S6K, β-actin, phospho-S6K, phospho-P70S6K and secondary horse radish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse antibodies were purchased from Cell Signal Technology (Danvers, MA, USA).
Cell lines and cell culture
Huh7, one of human HCC cell lines, was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Huh7R, an acquired sorafenib-resistant HCC cell line, was established from Huh7 cells as follows: When the cells were in the logarithmic growth phase, changed the culture medium, added a lower concentration of sorafenib to it for 24 h, then performed cell inheritance, and repeatedly stimulated with this concentration until it was stable. When the sorafenib concentration reached 4-5 times the IC50 value of the sensitive strain, the resistant strain was obtained. Two cells were cultured in RPMI-1640 (Hyclone, Salt Lake City, UT, USA), supplemented with 15% fetal bovine serum (FBS) (Sijiqing Bioengineering Materials, Hangzhou, China) and incubated at 37 ℃ with 5% CO2.
Cell viability assay
Cells (10,000 cells per well) were seeded into 96-well plates, allowed to adhere overnight, and exposed to a range of different drug concentrations. After 12, 24, 48 and 72 h, 10 µL of 5 mg/ml MTT dissolved in PBS was added to each well and incubated for 4 h at 37 ℃. The medium was aspirated, and 100 µL of DMSO was added and shaken for 15 minutes to each well. Absorbance was measured at 490 nm. IC50 value was calculated by GraphPad Prism Version 5.0 software.
Cells were cultured into six-well plates at a density of 2×103 cells per well for 24 h and drug experiments previously designed were carried out. After two weeks, when cellular clones were clearly visible, they were fixed in 4% paraformaldehyde for 15 min, and then stained with 0.1% crystal violet for 30 min. Subsequently, 6-well plates were thoroughly washed with water to count the number of clones. Colony formation rate reflects two important characteristics of cell population dependence and proliferation ability. All experiments were done in triplicate.
Cells (2×105 cells/well) were plated overnight in 6-well plates with 15% FBS medium. All wells covered with cells were emerged scratched areas with a 10 µL tip. PBS was used to remove injured cells. Serum-free medium with various drug were added to corresponding well and incubated at 37 ℃ in a humidified atmosphere with 5% CO2. Pictures with a light microscope were used to record the width of the scratched area after 24 and 48 h.
Cell cycle assay
Cells were digested by 0.25% trypsin-EDTA after incubated with different drug combinations for 24h, washed by PBS three times and fixed through 70% precooled ethanol. Before testing, cells were rewashed by PBS, added 50 μg/ml propidium iodide and 100 μg/ml RNase A in the dark for 30 min at 37 ℃ followed by flow cytometry analysis (BD FACSCalibur, USA). The distribution of cells at specific cell cycle stages was assessed with ModFit Version 3.0 software (Verity Software House, Topsham, ME).
After drug treatment for 24h, cells from different experimental groups were collected. After washed with cold PBS, cells were resuspended in 500 µL binding buffer containing 5 µL PI and 10 µL FITC-labelled Annexin-V and incubated for 15 min in the dark at room temperature. Cell apoptosis was detected using Flow cytometry analysis (BD Biosciences), and FlowJo Version 7.6.1 software (FlowJo, Ashland, OR) was operated to analyze apoptotic rate.
Whole cell lysates were obtained from the combination of Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, Jiangsu, China) and a mixture of protease and phosphatase inhibitor (Beyotime, Jiangsu, China). The BCA protein assay kit (Biosharp, Hefei, China) was applied to determine protein concentration. Supernatants per sample were aliquoted, mixed with loading buffer, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene Fluoride (PVDF) membrane. After blocked with 5% skim-milk, corresponding primary antibodies were employed to react at 4 ℃ for 24 h, then the secondary antibody binding reaction time was set to 1 h at 37 ℃. Clarity Western ECL substrate (Bio-Rad, Hercules, CA) was assisted to visualize the signals. We quantified protein expression using Image J Version 1.48 software (NIH, Bethesda, MD) and regarded β-actin as a loading control. Relative expression of indicated proteins were normalized to β-actin.
All in vitro assay results represent three replicates of three independent experiments performed under the same conditions. All data were expressed as mean values ± standard deviation. Statistical tests were performed on the paired values using the Student's t-test (p <0.05) was considered statistically significant).