Cell lines and reagents
Normal human hepatocytes THLE-2 and HHL-5 and human HCC cell lines Huh7, HepG2, and SK-Hep1 were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 (Hyclone; Salt Lake City UT, USA) and 10% fetal bovine serum (FBS) and maintained at 37 °C with 5% CO2. IGF-1 was purchased from Peprotech (Shanghai, China), sorafenib (#HY-10201S2) from Medchem Express (Monmouth Junction, NJ, USA), and podophyllotoxin (PPP) from Sigma Aldrich (St. Louis, Missouri, USA). The solvent/vehicle for PPP, IGF-1, and Sorafenib was ultrapure water, and the samples were stored at -20 °C. The lentivirus vector was from Sangonbiotech Genechem (Shanghai, China). IGF-1R, phospho-IGF-1R(p-IGF-1R), Akt, phospho-Akt (Ser473) β-actin, mTOR, phospho- mTOR (Ser2481), MEK1/2, phospho-MEK1/2, ERK1/2, phospho-ERK1/2, cleaved-caspase3, caspase3, poly ADP-ribose polymerase (PARP), cleaved PARP, horseradish peroxidase-coupled goat anti-rabbit and anti-mouse antibodies were purchased from Cell Signal Technology (Danvers, MA, USA). EdU and CCK-8 were purchased from Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). JC-1 was obtained from Sigma-Aldrich (St. Louis, MO, USA). Hoechst 33,258 and Alexa fluor® 488 phalloidin were purchased from Labgic Technology Co., Ltd. (Biosharp, Hefei, China).
Cell scratch healing experiment
Cells in exponential growth period were collected after trypsin digestion and diluted to 2.5 × 105 cells/mL. Six-well plates, marked on the back and containing 2 mL of cell suspension per well, were cultured at 37℃ and 5% CO2. After 24 h, the plates were scratched with a 10µL pipet tip held perpendicular to a horizontal line on the bottom surface. The plates were washed with PBS buffer 3 times to remove cell debris. Two milliliters of serum-free medium and corresponding drugs for treatment were added for various times. Wound size ratio % (24 or 48 h) = Wound size (0 h) - Wound size (24 or 48 h) / Wound size (0 h). Photographs were taken under an optical microscope, and cell mobility was calculated.
Cell clone formation experiment
Cells in logarithmic growth stage were digested with trypsin and blown into single cells. Cell suspensions were inoculated in six-well plates at a density of 1000 cells per well and placed in a cell incubator for 10 days. Medium was removed from the plates, and the plates were washed with PBS 3 times. The cells were fixed in 4% paraformaldehyde for 20 min, washed with PBS 3 times, dyed with 0.1% crystal violet dye for 10 min, photographed, and counted.
EdU cell proliferation experiment
Cells at 2 × 105 cells per well were cultured in 24-well plates overnight. Cells in each well were treated with corresponding drugs for 24 h and incubated for 2 h after 10 µL of EdU working solution were added to each well. The culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 15 min. The cells were washed with PBS 3 times, incubated with Triton X-100 at room temperature for 15 min, and washed with PBS 3 times. Click reaction solution was added to cover the sample evenly and incubated at room temperature for 30 min. Photographs were taken under an inverted fluorescence microscope, and the cell proliferation rate was calculated.
1.5 × 105 cells per well were added to 24-well plates and cultured overnight. The culture medium was removed, and a mixture of 250 µL culture medium and lentivirus, with an MOI value of 30, was added to the wells. Incubation was continued for 4 h, and 250 µL culture medium was added. The next day, the medium containing virus was removed, fresh culture medium was added, and incubation was continued.
Cell migration was measured with Transwell chamber (JET BIOFIL, Guangzhou, China). 3 × 105 HCC cells, resuspended in 100 µL RPMI 1640, were inoculated into the upper chamber; 600 µL RPMI 1640, supplemented with 10% FBS, were added into the lower chamber. After 24 h of culture, the cells in the upper layer were wiped off with a wet cotton swab. The cells on the other side of the membrane were stained with crystal violet, photographed, and counted under a microscope.
Acridine orange/ethidium bromide staining
Acridine orange (AO) working solution, 100 µg/mL, (Solarbio, Beijing, China) and ethidium bromide (EB) (Keygene Biotech, Nanjing, China) were mixed in the ratio of 1:1, diluted 20 times with PBS, mixed evenly, made into working solution, and added dropwise to the cell climbing tablet. The morphology of apoptotic cells was analyzed and counted by inverted fluorescence microscope. The percentage of apoptotic cells = (total number of apoptotic cells / total number of cells counted) × 100%。.
After the cells had adhered to the wall overnight, they were fixed with methanol for 30 min and washed with PBS. 200 µL IGF-1R specific antibody were added and incubated overnight at 4℃. The slides were washed with PBS, and Alexa fluor ® 488 phalloidin, 200 µL, were added and incubated in the dark for 30 min, and the slides were washed with PBS 3 times. Hoechst 33,258 was added and reacted for 10 min in the dark. The slides were washed with PBS and photographed under an inverted fluorescence microscope.
After incubation for 24 h, the cells were fixed with methanol and washed with PBS. Peroxidase blocker was added dropwise and incubated at 37℃ for 15 min. The slides were washed with PBS 3 times, and nonimmune animal serum for blocking of nonimmune proteins was added at 37℃ for 15 min. The serum was discarded, and 200 µL of IGF-1R specific antibody was added dropwise and incubated overnight at 4℃. After PBS wash, 200 µL of secondary antibody was added and incubated at 37℃ for 15 min; after PBS washing 3 times, 100 µL of horseradish-labeled streptomycin working solution was added. After incubation at 37℃ for 15 min and PBS wash 3 times, freshly prepared diaminobenzidine chromogenic solution was added, and the slides were counterstained with hematoxylin, rinsed with running water, and photographed under the upright fluorescence microscope.
Mitochondrial membrane potential detection
1.5 × 105 cells were placed in each well in the 24-well plates and cultured overnight. After drug treatment, 200 µL of JC-1 working solution was added to each well and incubated for 20 min. After PBS wash 3 times, Hoechst 33,342 staining was added and reacted away from light for 10 min. After cleaning with PBS, photographs were taken under inverted fluorescence microscope. In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form a polymer, which fluoresces strongly red; in unhealthy mitochondria, due to the decrease or loss of membrane potential, JC-1 exists only as a monomer in the cytoplasm, which fluoresces green.
Cells were treated with various drugs, and cells were collected to extract proteins. Separation gel was prepared and concentrated, and protein was loaded for electrophoresis. The separation gel was removed, and the membrane (Millipore, USA) was transferred for protein assay. The membrane was blocked with 5% skim milk for 1 h, then placed in the primary antibody solution at 4 ℃ overnight. The membrane was washed with TBST for 3 times. The bands were incubated with HRP-conjugated secondary antibody for 30 min, and the gray value was analyzed by Image J version 1.48 software.
Cell counting kit-8 experiment
1.5 × 103 cells per well were inoculated in 96-well plates overnight. After 24 h of drug treatment, 10 µL CCK-8 solution was added to each well and incubated in the cell incubator for 2 h. The absorbance was measured at 450 nm, and the cell viability was calculated by Graphpad Prism version 5.0 software. (Graphpad, La Jolla, CA, USA).
The animal experiments were approved by the ethics committee of Medical College of Anhui University of technology. 100 µL SK-Hep1, SK-Hep1 cells transfected with siRNA2-IGF-1R lentivirus (SK-Hep1siRNA2 − IGF−1R), and SK-Hep1 cells transfected with siRNACtrl-IGF-1R lentivirus (SK-Hep1siCtrl − IGF−1R) suspension (2 × 107 cells/ml) were injected subcutaneously into the right dorsum of 5-week-old female BALB/c nude mice (Vital River Laboratories, Beijing, China). The mice injected with SK-Hep1 cell suspension were randomly divided into 3 groups: (1) control group, 100 µL normal saline per day; (2) sorafenib group, sorafenib 30 mg/kg per day; (3) PPP + sorafenib group, PPP 10 mg/kg + sorafenib 30 mg/kg per day. SK-Hep1siRNA2 − IGF−1R and SK-Hep1siCtrl − IGF−1R tumor-bearing mice were fed 30 mg/kg sorafenib daily. Each group contained five mice. The solvent for PPP, IGF-1, and sorafenib was saline, and Ctrl groups were injected with the same amount of saline. Each group of mice received treatment for 3 weeks, and the tumor size and body weight were measured every 3 days. Tumor volume was calculated according to the formula V = L×W2 × 1/2 (V, volume; L, tumor length; W, tumor width).
SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) and Graphpad Prism 5.0 software were used for data analysis. All experiments were repeated at least 3 times, and the results were expressed as mean ± standard deviation (SD). One-way ANOVA was used to test the differences between groups. p < 0.05 was statistically significant.