Regents
Sorafenib (S125098) and Metformin (M107827) were obtained from Aladdin Reagent Company. MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazoliumbromide) was purchased form Biofroxx (Einhausen, Germany) and dissolved in 0.01M phosphate-buffered saline (PBS).
Antibodies against MLKL, cleaved-Caspase3 were products of Bioworld Technology (USA) and antibodies against NHE1, GAPDH, c-Myc, CyclinD, and β-Actin were products from Santa Cruz Biotechnology (USA). Antibodies against α-Tubulin, N-cadherin, E-cadherin, MMP-9, and MMP-2 were obtained from Proteintech Group. For the secondary antibodies, Mouse IgG-HRP and rabbit IgG-HRB secondary antibodies were purchased from Santa Cruz Biotechnology.
Cell culture
Human HCC cell lines (HepG2, MHCC-97H, SMMC-7721, and LM3) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). XB-1 cells were extracted from HCC patients. All cell lines were cultured in Dulbecco’s Modified Eagle Medium (Gibco, Carlsbad, CA, USA) and were supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco, Paisley, Scotland), 100U/ml streptomycin and 100 U/ml penicillin at 37°C with 5% CO2.
The cells were maintained in a in a humidified incubator containing 20% O2, 5% CO2, and 75% N2 as normoxia. For the hypoxic condition, the cells were exposed to a gas mixture containing 2% O2, 93% N2, and 5% CO2 in a humidified atmosphere at 37°C.
Clinical samples
Fresh primary liver cancer tissues, normal liver tissues, and para-carcinoma tissues were obtained from HCC patients at Nanjing Drum Tower Hospital, China.
Combined effect evaluation
CompuSyn software calculated Dm, m, and CI values based on the mathematical model. The drug interaction can be quantitatively determined by the size of the CI value. The combination index (CI) was calculated as follows:
CI = (D)1/(DX)1 + (D)2/(DX)2
where (DX)1 and (DX)2 are the doses of metformin and sorafenib in combination, and (D)1 and (D)2 are the doses of each drug alone that inhibits 50% cell growth. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively. Data analysis was performed with CalcuSyn software (Biosoft, Oxford, UK).
Animal Studies
5-6-week-old female BALB/c nude mice were purchased from SLAC laboratory (Shanghai, China) and maintained under specific pathogen-free conditions in controlled lighting conditions (12 h light/day) with standard laboratory food and water.
To establish the subcutaneous transplantation model, 2.5 × 106 XB-1 cells were subcutaneously injected into the flanks of the mice. Once the tumor volume reached 100mm3, the mice were randomly divided into four groups with five individuals per group based on tumor size. The treatment groups received oral administration of sorafenib (60mg/kg) every two days and metformin (200mg/kg) twice a week for three weeks. The tumor xenografts were then dissected and measured, and tumor volume (TV) was calculated using the formula: TV (mm3) = D/2 × d2, where D is the longest diameter and d is the shortest diameter.
A lung metastasis model was established in BALB/c nude mice by intravenous injection with 3 × 106 XB-1 cells. Two days post injection, the mice were orally treated either with vehicle solution (0.9% NaCl) every two days, 60 mg/kg sorafenib (0.9% NaCl) every two days, 200 mg/kg metformin (0.9% NaCl) twice a week, or 60 mg/kg sorafenib in combination with 200 mg/kg metformin.
To establish an orthotopic recurrence model, mice were anesthetized, and XB-1 cells were injected into the liver. Two weeks later, the second operation was carried out to remove the tumor tissue, and drug administration began after wound healing. The health status of the mice, tumor size, and tumor recurrence were recorded.
Western blotting analysis
Cells were washed with cold PBS and lysed in RIPA Lysis buffer (ThermoFisher, USA) containing protease/phosphatase inhibitors. After lysates concentration was determined by BCA protein assay (Pierce BCA Protein Assay Reagent - ThermoScientific), an equal number of denatured proteins was subjected to SDS-PAGE gel electrophoresis and then transferred onto a nitrocellulose membrane (BioTrace NT, PallCor, USA), which was then blocked by 5% non-fat milk in PBS, followed by incubation at 4°C with specific primary antibodies overnight. Then, membranes were incubated with HRP goat anti-rabbit immunoglobulin G (IgG; H + L) or anti-mouse IgG (H + L) secondary antibody (Biosharp) for one hour. Then the membrane was detected by ECL detection kits (Tanon).
Wound healing assay
Hepatocellular carcinoma cells were seeded into a six-well plate, and a pipe was used to wound the cells. Before giving the indicated treatment, wounded cells were washed lightly with phosphate-buffered saline (PBS). Then, the plates were incubated for 24 hours and migrated distances of different group cells were analyzed by microscope with five randomly chosen fields.
Transwell assay
The Transwell Boyden Chamber (8 µm, Millipore) with Matrigel (Corning) was used to evaluate cell invasion. Cells were suspended in serum-free DMEM and added to the upper chamber at a density of 3 × 105 cells per well. The upper chamber was then placed in a 12-well plate containing medium with 10% FBS. After incubation for 48 hours, cells that had invaded through the Matrigel and filter membrane were fixed with methanol and stained with 0.1% crystal violet. Three randomly selected fields were imaged and analyzed for each group.
Flow cytometry analysis
Cells were harvested and stained with the Annexin V/PI Cell Apoptosis Detection Kit (Vazyme, China) according to the manufacturer’s instructions. Data were analyzed by FlowJo version 10. All the bar charts were analyzed by GraphPad Prism 8 software (GraphPad Software, San Diego, CA).
Immunohistochemistry(IHC)
Paraffin embedding sections were heated at 60°C for half an hour and dewaxed. For the IHC assay, tissue sections were incubated with 0.3% Triton-X 100 for 20 minutes after antigen retrieval. Then, the solution containing goat serum was used to block nonspecific binding sites. Next, primary antibodies were added and incubated at 4°C overnight. Before interacting with the DAB solution, tissues were incubated with biotin-labeled secondary antibodies at room temperature for 30 minutes. Finally, tissues were stained with hematoxylin and covered by neutral gum, all the process was performed with standard techniques. The immunohistochemistry kit was purchased from Shanghai Yeasen BioTechnologies.
Statistical analyses
The data shown in the study were expressed as mean ± SD from at least 3 independent experiments, each in triplicate samples for individual treatment or dosage. Statistical analyses were performed using ANOVA coupled with a post hoc test.