The HepG2 cells were provided by the Institute of Modern Physics, Chinese Academy of Science (Lanzhou, China). DMEM was purchased from Hyclone; GE Healthcare Life Sciences (Logan, UT, USA); fetal bovine serum (FBS) was purchased from Sijiqing Company (Hangzhou, China). MTT was purchased from Amresco, LLC (Solon, OH, USA), BSI-201 was purchased from Sigma; Merck KGaA (Darmstadt, Germany). AZD2281 and AG014699 were purchased from Selleck Chemicals (Shanghai, China). The primary antibodies (GAPDH, Caspase 3, Caspase 8, Bcl-2, Bax, PTEN, TIMP 3 and MMP3) were purchased from Affinity Biologicals Inc. (Ancaster, ON, Canada). The secondary antibody was purchased from Beyotime Institute of Biotechnology (Haimen, China).

The HepG2 cells were cultured in DMEM medium containing 10% FBS, and 100 U/ml antibiotics (penicillin:streptomycin, 1:1) was added. The cells were incubated at 37°C in an incubator with 5% CO₂ and 95–98% relative humidity. Cells at the logarithmic phase were treated with 0.25% trypsin and passaged.

The HepG2 cells were cultured at a density of 5×10³ /ml in 96-well plates, and divided into the following control and drug groups: Blank group (complete medium); control group (complete medium containing <1% DMSO); AG014699 group (5, 10, 20, 40 and 80 µmol/l); AZD2281 group (50, 100, 200, 400 and 800 µmol/l); BSI-201 group (10, 20, 40, 60 and 80 µmol/l). Each group included six wells. Subsequently, 20 µl MTT was added to each well following 24, 48, 72 and 96 h of culture. After 4 h, the culture medium was aspirated and 150 µl DMSO was added to each well. The optical density (OD) was measured on an absorbance microplate reader with a wavelength of 570 nm. The inhibition rate was calculated according to the following formula: Inhibition ratio=1-(OD_{drug group}-OD_{control group})/(OD_{control group}-OD_{blank group}) ×100%.

The HepG2 cells at the logarithmic phase were seeded into 6-well plates at a density of 2.5×10⁴ /ml. The PARP-1 inhibitors AG014699 and BSI-201, which were sensitive to the HepG2 cells, were selected for the apoptotic assay. After 48 h, the cells were cultured with AG014699 at concentrations of 10, 30 and 50 µmol/l, or BSI-201 at concentrations of 20, 40 and 60 µmol/l for 48 h at 37°C, following which the cells were digested with 0.25% trypsin and washed twice with PBS. To 100 µl of the solution (1×10⁴ cells), 5 µl of annexin V-fluorescein isothiocyanate (BD GmbH, Heidelberg, Germany) and 10 µl propidium iodide (20 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) were added. Analysis was performed using a FACScan and CELLQuest software version 2.1.4.7 (BD Biosciences, Franklin Lakes, NJ, USA).

The HepG2 cells at the logarithmic phase were seeded into 6-well plates at a density of 5×10⁴ /ml. After 48 h, AG014699 (10, 30 and 50 µmol/l) or BSI-201 (20, 40 and 60 µmol/l) were added then cultured for 48 h at 37°C. Cells were digested with 0.25% trypsin containing 0.02% EDTA. After 1 min, the cells were gently agitated with 1.5 ml of PBS buffer. After centrifugation at 55 × g at 4°C for 5 min, the supernatant was removed and washed three times with PBS. Then 200 µl of PMSF and RIPA lysate (1:100) added for 30 min. The lysate was slowly agitated with a 2 ml syringe and allowed to lyse sufficiently. The centrifuge was preheated to 4°C and the lysate centrifuged at 8,051 × g for 15 min. The supernatant was dispensed into a 100 µl centrifuge tube and the protein concentration was measured using a Pierce BCA Protein Quantitative Assay kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sample proteins were separated by electrophoresis on a 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride. Membranes were blocked with 5% non-fat milk powder in TBST buffer (Tris-buffered saline, 0.05% Tween 20) and incubated at 4°C for 2 h with primary antibodies against Caspase 3 (AF835; 1:1,000), Caspase 8 (AF705; 1:1,000), Bax (AF820; 1:1,000), Bcl-2 (AF810; 1:1,000), PTEN (AF847; 1:1,000), TIMP3 (AF0265; 1:1,000) and MMP3 (AF548; 1:1,000; all from Affinity Biologicals Inc.). The membranes were washed three times in TBST and then incubated for 2 h at room temperature with the secondary antibody (horseradish peroxidase-labeled goat anti-rabbit; s0001; 1:1,000; Beyotime Institute of Biotechnology, Haimen, China) and then washed three times in TBST. The gray value of each band was analyzed using ImageJ software, version 2.1.4.7 (National Institutes of Health, Bethesda, MD, USA).

The HepG2 cells at the logarithmic growth phase were starved for 12 h, and then digested using trypsin. The cells, in DMEM containing 5% FBS, were seeded into a Transwell chamber at a density of 5×10⁵ cells/ml. The chamber was placed into 24-well plates, and placed in an incubator at 37°C, 5% CO₂ overnight. Following incubation, the medium in the chamber was removed, and AG014699 or BSI-201 were added with final concentrations of 5 and 10 µmol/l, respectively. The control group contained 300 µl serum-free medium with <1% DMSO. The lower chamber contained 600 µl DMEM containing 25% FBS. The cells were placed into the cell incubator and cultured for 48 h at 37°C. The medium was then removed and the cells were washed twice in PBS buffer, followed by fixing in 4% formaldehyde for 3–5 min at room temperature and treatment with methanol for 20 min. Subsequently, the cells were washed twice with PBS, and the dry Transwell chamber was placed into the lower chamber for staining with crystal violet for 15 min. The cells were washed twice in PBS and visualized using an inverted microscope to examine cell migration.

The results are expressed as the mean ± standard deviation. SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used to analyze data. An independent t-test was used to determine the differences between two groups. One-way analysis of variance was used to analyze the differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

The results showed that different concentrations of the PARP-1 inhibitors inhibited the proliferation of HepG2 cells. As concentrations increased, the inhibitory effect was enhanced (Fig. 1). After 24 h, the half maximal inhibitory concentrations of AG014699, BSI-201 and AZD2281 were 80, 160 and 800 µmol/l, respectively. After 48 h, the half maximal inhibitory concentrations of AG014699, BSI-201, AZD2281 were 20, 30 and 400 µmol/l, and at 96 h, the half maximal inhibitory concentrations were 10, 25 and 300 µmol/l at 72 h, and 5, 20 and 200 µmol/l.

According to the results of the MTT assay, the two PARP-1 inhibitors, AG014699 and BSI-201, were selected as they exhibited sensitive inhibitory effects on the HepG2 cells. The results showed that the apoptotic rates of the HepG2 cells increased with increasing concentrations of these two inhibitors. There were significant differences between the groups (P<0.01; Fig. 2). Similarly, in the HepG2 cells treated with 20, 40 or 60 µmol/l BSI-201 for 48 h, the apoptotic rates of the HepG2 cells also increased, and significant differences were found between the groups (P<0.01; Fig. 3).

Following treatment of cells for 48 h with AG014699 at concentrations of 10, 30 and 50 µmol/l, the protein levels of Caspase 3, Caspase 8 and Bax were higher, compared with those in the control group (Fig. 4A and B), which was also the case in the cells treated with BSI-201 concentrations of 20, 40 and 60 µmol/l (Fig. 4C and D). The protein levels of Bcl-2 in the two treatment groups were reduced, compared with those in the control groups (Fig. 4).

Following treatment of the HepG2 cells for 48 h with 10 µmol/l of BSI-201 or 5 µmol/l AG01469, the results showed that the numbers of cells, which migrated into the lower chamber were lower, compared with those in the control groups, and this difference was significant (P<0.01; Fig. 5). When the HepG2 cells were treated with AG014699 at concentrations of 10, 30 and 50 µmol/l (Fig. 6A and B) or with BSI-201 at concentrations of 20, 40 and 60 µmol/l (Fig. 6C and D) for 48 h, the protein levels of PTEN and TIMP3 were higher, compared with those in the control group, whereas the levels of MMP3 were lower, compared with those in the control group.