Cell lines and chemicals
Two human hepatocellular carcinoma-derived cell lines were used: (a) Huh7 cells expressing mutant p53 (tyrosine → cysteine at codon 220, Y220C) ; (b) Hep3B cells lacking p53 . Huh7 and Hep3B cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Triptolide (purity, ≥ 97%; molecular weight, 360.40096) was purchased from Shanghai Tongtian Biotechnology Co., Ltd. (Shanghai, China). Tempol was bought from MedChemExpress (MCE) (Shanghai, China).
Cell culture and transfection of plasmids
HuH7 and Hep3B cells were maintained in High Glucose Dulbecco's modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/ml penicillin and 100 ug/ml streptomycin (Gibco).
The mt p53 (Y220C) ORF clone of Homo sapiens tumor protein p53 which was used as a p53 mutant (Y220C) expression plasmid and the vector control were purchased from OriGene (Rockville, MD, USA). Lipofectamine 3000 kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was used for transfection of plasmids into Hep3B cells. Based on the manufacture’s instruction, Hep3B cells at approximately 80% confluence were transfected with mt p53 (Y220C) ORF clone expression plasmid or vector control in duplicate in a 6-well plate. After incubation in the atmosphere contained 5% CO2 for 8 h at 37 ℃ in a humidified incubator, the medium of a copy of transfected cells was replaced with fresh medium in which triptolide was added. After 40 h of culture, cells from each well were harvested for protein/mRNA expression or apoptosis induction analysis.
Approximately 2 000 cells per well were seeded in 96-well cell culture plates and incubated overnight in 100 microliter cell culture medium. Cells were subsequently treated with four different concentrations of triptolide for 48 h. Then, 10 microliter cell counting kit 8 (CCK-8) (MCE, Shanghai, China) was added into each well and the cells were incubated for another 3 h in the cell incubator. A microplate reader (BioRad, CA, USA) was used for measuring the absorbance at wavelength of 450 nm.
Annexin V-FITC/PI analysis
To determine the apoptosis and necrosis, cells were firstly incubated with various concentrations of triptolide for approximately 32 h and then were processed according to the instruction manual of FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA). Finally, the stained cells were immediately analyzed by a flow cytometer LSR Fortessa (BD Biosciences, San Jose, CA, USA) using the excitation wavelengths of 488 nm and 535 nm. At least 10 000 cells were used for analysis in each group.
Quantitative real-time PCR
The total RNA of cells was extracted with RNAiso Plus (TaKaRa, Otsu, Japan). The concentration and purity of RNA was measured by Nanodrop 2000c (Thermo Fisher Scientific, USA). cDNA was prepared using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). The primers for Real-time PCR analysis were listed in Table 1. The PCR amplification was completed on ABI PRISM 7900HT/FAST using TB Green Premix Ex Taq (Tli RNaseH Plus) (TaKaRa) and 40 cycles of 95 ℃ for 5 s and 60 ℃ for 30 s. GAPDH was used as an internal parameter. Fold change for the mRNA level of molecule in the treated groups versus that in the control group was calculated using the 2−ΔΔCt method [13, 27].
Cell cycle determination
Huh7 and Hep3B cells (1×106) were treated with triptolide (20 ng/ml) for 24 h. Cells were harvested and washed with ice-cold PBS. Cells were then gently resuspended in 5 ml of ice-cold ethanol (70ཞ80%) and stored at 4 ℃ for at least 18 h. After washing with PBS and staining buffer (BD Pharmingen) successively, the cells were incubated with PI/RNase staining buffer (BD Pharmingen) at room temperature for 15 min. Subsequently, the samples were determined by flow cytometry and cell cycle was analyzed with ModFit LT software (version 3.1) (Verity Software House, Topsham, ME, USA).
BCA Protein Assay kit (Pierce Chemical Co., Rockford, IL, USA) was used for evaluating the concentration of total protein. Forty microgram proteins of each group were separated by 4ཞ20% SurePAGE, Bis-Tris gels (GenScript, Nanjing, China); and transferred to polyvinylidene difluoride membrane (Merck Millipore, Billerica, USA); and then were probed with the specific primary antibodies and horseradish peroxidase-conjugated anti-IgG antibodies. Target proteins were displayed by enhanced chemiluminescence (ECL) kit (BeyoECL Plus) (Beyotime, Shanghai, China) and the signals were captured by chemiluminescence detection system (Clinx Science Instruments, Shanghai, China). Rabbit monoclonal antibody against caspase-8, caspase-9, caspase-3, PARP, p21, p53, Phospho-p53 (Serine20) or c-myc as well as peroxidase-conjugated anti-rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA), Rabbit multiclonal antibody against GAPDH was purchased from Bioker Biotechnology (Hangzhou, China). Densitometric calculations of protein bands were performed by using ImageJ software (NIH, Rockville Pike, Bethesda, Maryland, USA)
SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was employed for the statistical analysis. Values were presented as mean ± S. D. of at least three experiments. Statistical comparisons were conducted using one-way analysis of variance (ANOVA). The level of significance was set at P < 0.05 or P < 0.01.