p53 and β-actin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). PTE, DEN, N-acetylcysteine (NAC), PFTα and most of the chemicals and reagents used in this study were procured from Sigma-Aldrich (St. Louis, MO, USA).

Animals were treated in accordance with the guidelines approved by the Animal Care and Use Committee of the Fourth Military Medical University (Shaanxi, China). C57 mice were purchased from the Experimental Animal Center of the Fourth Military Medical University. Mice were kept in individual cages with free access to food and water and the environment was at constant temperature and humidity conditions on 12-h light/dark cycles.

Mice were acclimated for 1 week and then randomly divided into 7 groups (n=15): Ctrl, DEN, DEN+PTE (100 mg/kg), DEN+PTE (200 mg/kg), DEN+PTE (200 mg/kg)+PFTα, DEN+PTE (200 mg/kg)+LV-SOD2 and DEN+PTE (200 mg/kg)+NAC. Mice were injected with DEN plus CCl₄ to construct the HCC model. In brief, mice were intraperitonially injected with DEN (200 mg/kg, BW) once and 2 weeks later mice were given a CCl₄ (3 ml/kg) injection thrice a week for 6 consecutive weeks. In the groups of DEN+PTE (100 mg/kg), DEN+PTE (200 mg/kg), DEN+PTE (200 mg/kg)+PFTα, DEN+PTE (200 mg/kg)+LV-SOD2 and DEN+PTE (200 mg/kg)+NAC, mice were intraperitonially injected with PTE (100 or 200 mg/kg daily) and/or PFTα (10 mg/kg daily) or SOD2 lentivirus twice (on 2 consecutive days) for 2 weeks throughout the experimental procedure. Mice in the DEN+PTE (200 mg/kg)+LV-SOD group were treated with 0.4×10⁸ TU LV-SOD2 through an intravenous injection in the tail. Twenty weeks after the injection of DEN, mice were sacrificed after overnight fasting. According to the accepted criteria, the tumors were counted and measured (19). Blood samples were separated and stored for further biochemical determination. A section of the tumor tissue was fixed in 10% paraformaldehyde for TdT-mediated dUTP nick end labeling (TUNEL) staining. Another section of the tumor tissue was frozen and cut for ROS detection while another area of tumor tissue was homogenized in saline for the determination of caspase activity. The remaining tissues were stored at −20°C for the determination of mRNA and protein expression.

HepG2 cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (FBS; 10%), 100 U/ml penicillin and 100 U/ml streptomycin. Cells were incubated at 37°C in a humidified incubator containing 5% CO₂. For the experimental treatment, cells were incubated with 12.5–100 µM PTE in serum-free medium for 24 h.

The scramble or SOD2 lentivirus was transfected into cells to build cells stably expressing SOD2 according to the manufacturer's instructions. Subsequently, cells were treated with 100 µM PTE for 24 h.

After the treatment, cell viability and proliferation were measured. An MTT assay was conducted to evaluate cell viability. A CCK-8 assay was performed to assess cell proliferation according to the manufacturer's instructions (Sigma-Aldrich). The absorbances at 570 and 450 nm were measured, respectively. Results are presented as folds of the control.

Serum levels of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.

TUNEL staining was conducted to measure the apoptosis in liver tissues and cells using the In Situ Cell Death Detection kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. The total number of cells and the number of TUNEL-positive stained cells were counted by an independent researcher. At least 6 random fields were counted for each slide. Results are expressed as the percentage of apoptotic cells.

The activity of caspase-3 in homogenates of liver tissues and cells was determined using commercial assay kits (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions.

The ROS level was detected by DHE, a superoxide sensitive probe. In brief, frozen liver sections and cells were incubated with 10 µM DHE at 37°C for 30 min in the dark. After being washed twice, slides or dishes were observed under a confocal microscope (Olympus, Tokyo, Japan).

Total RNA was extracted from the tissue samples and cells using TRIzol reagent according to the manufacturer's instructions (Invitrogen). mRNA (500 ng) was reversely transcribed into cDNA using the First Strand cDNA synthesis kit (Takara Bio, Inc., Otsu, Japan). Target gene expression was quantified by a real-time polymerase chain reaction (PCR) system using SYBR-Green reagents (Takara Bio, Inc.) in a Bio-Rad Cycling Biosystem (Bio-Rad Laboratories, Inc., Hercules, CA, USA). β-actin was used as an internal control. Amplification conditions were as follows: an initial step at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 63°C for 30 sec and then extension at 72°C for 10 sec. The relative amount of RNA was quantified using the comparative threshold cycle (C_t) (2^−ΔΔCt) method.

Liver tissues and cells were lysed with cell lysis buffer (50 Mm Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 mM NaF, 1 mM Na₃VO₄, and a protease inhibitor cocktail) on ice for 30 min. After centrifugation at 20,000 × g for 20 min at 4°C, the protein content of the supernatants was determined by BCA assay kit (Pierce, Rockford, IL, USA). Subsequently, equal volumes of supernatants and 2X SDS loading buffer were mixed and boiled for 5 min. Samples containing equal amounts of protein were subjected to SDS-PAGE and then transferred onto an NC membrane. After blocking with non-fat milk for 1 h at room temperature, the membranes were incubated with indicated primary antibodies overnight at 4°C. After being washed four times, the membranes were incubated in the appropriate horseradish peroxidase-conjugated secondary antibodies at 37°C for 45 min. The protein bands were visualized using chemiluminescent reagents according to the manufacturer's instructions (Thermo Fisher Scientific) and quantified using an image analyzer Quantity One system (Bio-Rad Laboratories, Inc.).

Results are expressed as the mean ± SEM. All the experiments were performed at least 3 times. The results were analyzed using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA) by one-way ANOVA followed by an SNK-q test for multiple comparisons. P<0.05 was considered statistically significant.

To investigate the effect of PTE on tumor growth, an HCC mouse model was established using DEN/CCl₄ administration. In Table I, we showed that the incidence of tumorigenesis, the number of tumors and the maximum size of the tumors in the DEN-treated mice were 100%, 18.6±2.9 and 9.7±0.8 mm, respectively. The administration of PTE did not affect the incidence of tumorigenesis (Table I). However, PTE significantly decreased the number of tumors and the maximum size of the tumors in the DEN-treated mice. The results showed that in the 100 mg/kg PTE group, the number of tumors and the maximum size of the tumors were reduced to 13.2±2.3 and 5.8±0.7 mm, respectively (Table I). In the 200 mg/kg PTE group, the number of tumors and the maximum size of the tumors were reduced to 6.4±3.7 and 4.0±0.6 mm, respectively (Table I). In addition, the activities of LDH, ALT, AST and ALP were significantly increased by DEN/CCl₄ treatment which were inhibited by PTE in a dose-dependent manner (Table II). The results indicated that PTE administration protected against tumor growth and liver injury in the DEN-treated mice. Moreover, we examined the effect of PTE on cancer cell proliferation in HepG2 cell lines. Cells were incubated with 12.5–100 µM PTE for 24 h and then cell viability and proliferation were measured. In Fig. 1A and B, we showed that 25–100 µM PTE concentration-dependently decreased cell viability and proliferation in HepG2 cells. The results indicated that PTE inhibited the proliferation of HCC cells in vitro.

Furthermore, the effect of PTE on apoptosis in liver tumor tissues and cells was evaluated. As shown in Fig. 1C, the activity of caspase-3 in the DEN-treated mice was significantly increased with PTE administration. In the HepG2 cells, 25–100 µM PTE concentration-dependently increased caspase-3 activity (Fig. 1D). Compared with the DEN group, PTE administration markedly increased the percentage of apoptotic cells (Fig. 1E). PTE (25–100 µM) concentration-dependently increased the percentage of apoptotic cells in the HepG2 cells (Fig. 1F). The results indicated that PTE activated the mitochondrial apoptotic pathway and induced significant apoptosis in tumor tissues and cells.

In the next step, we examined the possible role of p53 in the inhibitory effect of PTE on HCC. As shown in Fig. 2A, in the DEN-treated mice, PTE administration dose-dependently increased p53 expression. In Fig. 2B, we showed that 50 and 100 µM PTE concentration-dependently increased the protein expression of p53. To elucidate the role of upregulation of the p53 PTE-induced inhibitory effect on HCC in vivo and in vitro, mice and cells were treated with PFTα, an inhibitor of p53. The results showed that PFTα significantly inhibited the decrease in the number of tumors and the maximum size of the tumors as well as the LDH, ALT, AST and ALP activities induced by PTE in the DEN-treated mice (Tables I and II). In the presence of PFTα, the PTE-induced decrease in cell viability and proliferation in HepG2 cells was significantly suppressed (Fig. 2C and D). Moreover, inhibition of p53 by PFTα suppressed the PTE-induced increase in apoptosis both in mice in vivo and cells in vitro. These results indicated that p53-mediated regulation of apoptosis was involved in the inhibitory effect of PTE on HCC tumor growth in vivo and on cell proliferation in vitro (Fig. 2E and F).

Next, we evaluated whether ROS generation mediated apoptosis induced by PTE in vivo and in vitro. In Fig. 3A, we showed that PTE significantly increased DHE staining in liver sections, indicating an increase in ROS production. PFTα, overexpression of SOD2 by lentivirus infection and NAC, a potent antioxidant, significantly inhibited PTE-induced ROS generation in the tumor tissues (Fig. 3A). Consistently, in the HepG2 cells, PTE resulted in a significant increase in ROS generation which was inhibited by PFTα, overexpression of SOD2 by lentivirus infection and NAC (Fig. 3B). Moreover, the role of ROS generation in the effect of PTE on tumor growth, cell proliferation and apoptosis was examined. As shown in Tables I and II, the PTE-induced decrease in the number of tumors and the maximum size of the tumors as well as LDH, ALT, AST and ALP activities were inhibited by LV-SOD2 and NAC. LV-SOD2 and NAC also inhibited the PTE-induced decrease of cell viability and proliferation in the HepG2 cells (Fig. 3C and D). Moreover, the PTE-mediated increase in apoptosis in tumor tissues and cells was inhibited by LV-SOD2 and NAC (Fig. 3E and F). These results indicated that p53-mediated ROS generation was involved in the inhibitory effect of PTE on HCC tumor growth in vivo and on cell proliferation in vitro.

Considering the important role of SOD2 in counteracting ROS, we evaluated the role of SOD2 in p53-mediated apoptosis induced by PTE. Since overexpression of SOD2 significantly inhibited PTE-induced suppression of tumor growth and cell proliferation, it was indicated that a decrease in SOD2 was involved in tumor growth and cell proliferation in HCC. However, whether the expression of SOD2 was regulated by p53 was unknown. In the next step, we evaluated the expression of SOD2 using real-time PCR. As shown in Fig. 4A and B, in both DEN-treated liver tissues and HepG2 cells, PTE resulted in a significant decrease in SOD2 expression. LV-SOD2 enhanced the expression of SOD2 to a level that was markedly higher than that of the DEN group or control cells (Fig. 4A and B). In the presence of PFTα, the PTE-induced decrease in SOD2 was significantly inhibited both in vivo and in vitro (Fig. 4A and B). However, NAC had no significant effect on SOD2 expression, indicating that downregulation of SOD2 was upstream of ROS generation (Fig. 4A and B). The results demonstrated that downregulation of SOD2 was a link between upregulation of p53 and increase in the ROS level, which activated mitochondrial apoptosis and resulted in inhibition of tumor growth and cell proliferation.