Cell lines
Human HCC cell lines HepG2 and Huh7 were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated at 37 °C in a humidified incubator with 5% CO2 and 95% air. To obtain the CD133+ and CD133− HepG2 and Huh7 cells, HepG2 and Huh7 cell lines were stained with mouse anti-human CD133/1 (AC133) conjugated with VioBright FITC (Miltenyi Biotec, Germany) for 30 min at 4 °C in dark. Subsequently, cells were sorted by fluorescent-activated cell sorting equipment (Beckman Coulter, USA).
Gain-of-function and Loss-of-function of OPA1
To knockdown the gene of OPA1, OPA1 small interfering RNA (OPA1 siRNA, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. To overexpress OPA1, the OPA1 eukaryotic expression plasmid was generated by cloning the open reading frame of the OPA1 gene into the pcDNA3.1 plasmid (Life Technologies, Carlsbad, CA, USA). For transfection, OPA1 siRNA (50 pmol/ml) or OPA1 plasmid (2 µg/ml) plasmid was transfected by using the Lipofectamine™ 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction.
Cell viability assay
Cells were inoculated onto the 96-well plate at a density of 5,000 cells/well. After overnight incubation, the medium was replaced with fresh medium containing different concentrations of cisplatin and silibinin (Sigma Aldrich, St. Louis, MO, USA) for 48 h. Subsequently, CCK-8 (10 µl) (Sigma Aldrich) was added to each well and incubated for 2 h at 37 °C. In each well, the absorbance was measured at 570 nm. Cell viability was calculated by using the following formula: Cell viability rate = OD value in drug administration group/ OD value in control group. Half maximal inhibitory concentration (IC50) of cisplatin was calculated according to the cell viability curve.
Combination index (CI) with cisplatin and resveratrol
CI was based on the results of cell viability assay and was calculated by using the following formula: CI = E(A + B)/(EA + EB-EA × EB). EA represents as the inhibitory rate caused by cisplatin; EB represents as the inhibitory rate caused by silibinin; E(A + B) represents as the inhibitory rate caused by combination treatment with cisplatin and silibinin. It was considered as simple addition of the two drugs when CI ranged from 0.85 to 1.15; It was considered as the synergistic effect of the two drugs when CI was greater than 1.15; It was considered as the antagonistic effect of the two drugs when CI was less than 0.85.
Separation of mitochondria fraction and cytosol fraction
Cells were inoculated onto the 6-well plate at a density of 5 × 105 cells/well. After overnight incubation, the medium was replaced with fresh medium containing cisplatin (5 µM) and silibinin (5 µM) for 48 h. Cells were then collected and washed with PBS. Subsequently, Mitochondria fraction and cytosol fraction of HCC cells were separated by using Mitochondria/Cytosol Fraction Kit (BioVision, USA) according to the manufacturer’s instruction. The obtained mitochondria fraction and cytosol fraction was directly used for the western blot assay.
Western blot analysis
Total proteins from HCC cells were extracted by using lysis buffer (Cell Signaling Technology, Danvers, MA, USA). 50 µg of the obtained proteins were separated on a 12.5% SDS-PAGE system as described [21]. Subsequently, proteins were transferred to PVDF membranes (Millipore, Billerica, MA, USA) in transfer buffer. After blocking with 5% non-fat dried milk for 2 h, the membranes were incubated with the primary antibodies. Antibodies of anti-Mfn2 and anti-OPA1 were purchased from Abcam (Cambridge, MA, USA); antibody of anti-Mfn1 was purchased from Sigma Aldrich. Antibodies of anti-cytochrome c, anti-caspase-9, anti-caspase-3 and anti-GAPDH were purchased from Cell Signaling Technologies. After incubation with primary antibodies overnight at 4 °C, the immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA).
Measurement of apoptotic rate
Cells were inoculated onto the 6-well plate at a density of 5 × 105 cells/well. After overnight incubation, the medium was replaced with fresh medium containing cisplatin (5 µM) and silibinin (5 µM) for 48 h. Cells were then collected and washed with PBS. Subsequently, cell apoptosis was measured by using Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich) according to the manufacturer’s instruction. Annexin V positive cells were calculated as the apoptotic cells.
Statistical analysis
Data (mean ± standard deviation) were statistically analyzed by using one-way analysis of variance (ANOVA) and Bonferroni’s post hoc test through SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered to indicate a statistically significant.