HepG2 and QSG7701 liver cancer cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were maintained in Dulbecco's modified eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Cell culture was carried out in a standard cell culture medium containing L-glutamine (2 mM), streptomycin (100 µg/ml) and penicillin (100 U/ml) in an incubator with humidified 5% CO₂ and 95% air atmosphere. Triptolide was obtained from Sigma-Aldrich (St. Louis, MO, USA) and its stock solution in dimethyl sulfoxide (DMSO) was stored at −10°C. Pifithrin-α was obtained from Calbiochem (La Jolla, CA, USA).

Viability of HepG2 and QSG7701 cells was determined using a CCK-8 kit (Dojindo Molecular Technologies Inc., Rockville, MD, USA). HepG2 and QSG7701 cells were seeded at a density of 2×10⁶ cells/well into 96-well plates and treated with 10–100 µM concentrations of triptolide for 48 h. After incubation, the CCK-8 solution (10 µl) was added to each well of the plate and the plates were incubated in incubator at 37°C for 3 h. Absorbance was recorded three times independently using a microplate reader (Bio-Rad Laboratories, Richmond, CA, USA) at 450 nm.

HepG2 cells put into the tissue culture slides were allowed to undergo attachment for 48 h. The cells were then treated with various concentrations of triptolide for 48 h, washed with phosphate-buffered saline (PBS) and subsequently put into the 1X binding buffer (500 µl). A total of 5 µl each of Annexin V-FITC and propidium iodide were added into the plates and the cells were incubated under dark conditions for 5 min. The plates were analyzed using flow cytometry (BD FACSCalibur; BD Biosciences, San Jose, CA, USA) to determine the apoptotic cells.

HepG2 cells after incubation with various concentrations of triptolide were harvested and then were washed with ice-cold PBS. The cells were treated with RIPA lysis buffer (Roche Diagnostics, Shanghai, China) under ice-cold conditions for 45 min. The lysates were centrifuged at 12,000 rpm for 15 min at 4°C to collect the supernatant. Bradford method was used for the determination of the protein concentration in lysate. The proteins were separated by electrophoresis using 8% SDS-polyacrylamide gel and subsequently were transferred to NC membrane (Millipore, Billerica, MA, USA). The membrane incubation was performed with 10% dry milk in TBST to block non-specific binding sites. The membrane incubation with primary antibodies against Bax, DR5, p53, p53, histone-H2A.X and p-H2A.X (Cell Signaling Technology, Danvers, MA, USA) was performed overnight. The membranes were then washed with PBS and were incubated with secondary antibodies for 1 h. Expression of the desired proteins was examined using an ECL kit (Millipore) according to the instructions from the manufacturer. The bands were visualized using autoradiography with Hyperfilm (Millipore).

Into the culture plates (100 mm diameter) HepG2 cells were seeded at a density of 2×10⁶/plate and grown to confluence. The cells were then treated with triptolide for different durations. The cells were collected, PBS washed and subjected to fractionation into separate nuclear and cytosolic fractions using kit from Qiagen (Valencia, CA, USA). The sample purity of the nucleus and cytosol was checked using Lamin B and GAPDH as markers, respectively.

HepG2 cells seeded into the plates were treated with triptolide for analysis of DNA binding potential of p53. The nuclear fractions obtained as mentioned above were examined using the TransAM p53 kit according to the protocol provided by the manufacturer (Active Motif, Carlsbad, CA, USA).

Following treatment with triptolide for different durations HepG2 cells were collected to isolate total cell RNA using Isogen (Nippon Gene, Co., Ltd., Tokyo, Japan). Incubation of the RNA with DNase I was carried out to digest DNA that was contaminated. The RNA samples were purified by extraction using phenol and precipitation in ethyl alcohol. Ready-To-Go RT-PCR Beads (Amersham Pharmacia Biotech, Uppsala, Sweden) were used for the preparation of cDNA. AmpliTaq Gold^® DNA Polymerase, LD (Applied Biosystems, Foster City, CA, USA) was employed for CR of cDNA using the primers: p21, 5′-TTAGCAGCGGAACAAGGAGT-3′ and 5′-ATTCAGCATTGTGGGAGGAG-3′ upstream and downstream, respectively); Bax, 5′-ATCCAGGATCGAGCAGGGCG-3′ and 5′-GGTTCTGATCAGTTCCGGCA-3′; DR5, 5′-AAGACCCTTGTGCTCGTTG-3′ and 5′-TCACCTGAATCACACCTGG-3′; GAPDH, 5′-TGAAGGTCGGAGTCAACGGATTTGGGT-3′ and 5′-CATGTGGGCCATGAGGTCCACCAC-3′. Proteins were treated at 90°C for 15 min followed by amplification. Amplification of the cDNA was performed by the sequence of 94°C for 50 sec, 55°C for 50 sec and 72°C for 50 sec. Quenching of the reaction was done using elongation step at 75°C for 5 min. The 2% agarose gel and ethidium stain was used for the analysis of PCR products.

HepG2 cells were seeded at a density of 2×10⁶ into 60-mm cultural dishes (Falcon) and allowed to attain confluence. The cells were then treated with triptolide for 48 h. Following incubation, the cells were collected, washed with PBS and lysed. The Cell Death Detection ELISA Plus kit (Roche Diagnostics, Indianapolis, IN, USA) was used for the measurement of DNA degradation into the nucleosomal fragments.

HepG2 cells seeded at a density of 2×10⁶ into 60-mm cultural dishes were subjected to incubation with RNA for silencing p53 in transfection reagent (Mirus Bio, Madison, WI, USA). The plates were incubated for 48 h and then treated with triptolide for 48 h. After incubation, apoptosis induction in HepG2 cell cultures was analyzed using ELISA assay. Western blot assay was used to examine the expression of various proteins.

The data presented are the means ± standard error or division of the mean (SEM or SD). Student's t-tests and two-way ANOVA were used for the analysis of the data using the statistical program in GraphPad Prism Version 4.0 (GraphPad Software, Inc., San Diego, CA, USA). All experiments were performed in triplicate. A P<0.05 was taken to indicate statistically significant difference.

In HepG2 and QSG7701 cells treatment with various doses of triptolide for 48 h caused a concentration-dependent proliferation reduction. Treatment with range of triptolide concentrations from 10 to 100 µM led to a significant viability reduction at 50 µM of 48 h treatment in both HepG2 and QSG7701 cell lines (Fig. 1).

Annexin V-FITC staining showed that triptolide treatment of HepG2 cells for 48 h induced apoptosis. HepG2 cells were treated with 10–50 µM concentrations of triptolide for 48 h and then were analyzed by fluorescent microscopy. Triptolide treatment of HepG2 cells at 50 µM concentrations induced apoptosis in 56.45% cells compared to only 2.36% cells in the control cultures (Fig. 2).

Treatment of HepG2 cells with triptolide at various concentrations induced phosphorylation of p53 at serine-15 residue. The phosphorylation of p53 was significant at 2 h of treatment with 50 µM concentration of triptolide in HepG2 cells (Fig. 3).

Analysis of the phosphorylation of histone H2A.X at serine 139, indicator of DNA damage showed a significant increase by triptolide treatment from 12 h in HepG2 cells. However, no significant increase in the phosphorylation of histone H2A.X was observed at 1 h of triptolide treatment (Fig. 4).

Treatment of HepG2 cells with triptolide caused a significant increase in the level of p53 in HepG2 cell nuclei (Fig. 5). In 6 h-treatment with 0, 10, 20, 30, 40 and 50 µM concentration of triptolide the level of nuclear p53 was 15.3, 19.6, 28.5, 43.7, 63.8 and 91.5%, respectively (Fig. 5).

Treatment of HepG2 cells with triptolide at 50 µM concentration caused a significant increase in the binding potential of p53 to DNA. The binding potential of p53 to DNA was significantly higher in 6 h triptolide treatment (Fig. 6).

Triptolide treatment of HepG2 cells caused a significant increase in the expression of various genes such as p21, Bax and DR5 at 6 h. Compared to control HepG2 cells the expression of p21, Bax and DR5 genes was markedly higher on treatment with 50 µM concentration of triptolide (Fig. 7). In addition, triptolide treatment also increased the expression of miR-34b and miR-34c in HepG2 cells markedly (Fig. 7). However, no increase was observed in the expression levels of miR-34a in HepG2 cells on triptolide treatment (Fig. 7).

Treatment of HepG2 cells with p53 inhibitor, pifithrin-α prior to incubation with triptolide significantly prevented induction of cell apoptosis (Fig. 8). Suppression of p53 expression by siRNA inhibited the expression of p53 as well as its target genes along with the prevention of apoptosis induction (Fig. 8).