The present study was conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Tianjin Medical University General Hospital (Tianjin, China). This study was approved by the Ethics Committee of Tianjin Medical University General Hospital. All surgery and euthanasia were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

HepG2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere containing 5% CO₂.

HepG2 cells were cultured in 6-well plates until they reached 90% confluence and the medium was then removed. Sequences were cloned into vectors as previously described (24). Subsequently, HepG2 cells (1×10⁶) were transfected with pedue12.4-PI3K (100 nM), pedue12.4-TIPE-2 (100 nM) or pedue12.4-vector (100 nM) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h at 37°C. Cells that exhibited stable PI3K or TIPE-2 overexpression were selected according to the dihydrofolate reductase/glutamine synthetase screening system (Invitrogen; Thermo Fisher Scientific, Inc.).

HepG2 cells (5×10³) were incubated with TIPE-2 (0.5–2.5 mg/ml; SinoGenoMax Co., Ltd., Beijing, China) in 96-well plates for 24, 48 and 72 h at 37°C in triplicate for each condition. In the control group, cells were treated with 2 ml PBS instead of TIPE-2. Cell proliferation was determined using a MTT assay kit (Roche Diagnostics, Indianapolis, IN, USA). At each time point, 20 µl MTT (5 mg/ml) in PBS was added to each well and the plate was incubated for 4 h at 37°C. Most of the medium was then removed and 100 µl dimethyl sulfoxide was added to the wells to solubilize the crystals. Optical density was measured using a Bio-Rad ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at a wavelength of 450 nm.

Culture medium supplemented with TIPE-2 (2 mg/ml) or with PBS was added to 6-well plates (2 ml per well). HepG2 cells (1×10⁵ cells/well) were seeded into 6-well plates. Medium was replaced at 48 h following cell attachment and subsequently every 48 h. Cells were trypsinized (1% trypsin), suspended in a 500 µl volume of medium and a hemacytometer was used to determine the total number of cells per well on day 4. The colony formation assays were performed in triplicate.

Total cellular RNA was extracted from PI3K-overexpressed (PI3KOR), TIPE-2-overexpressed (TIPE-2OR) or control HepG2 cells using the RNeasy mini kit (Qiagen Sciences, Inc., Gaithersburg, MD, USA) according to the manufacturer's protocol. A total of 1 µg total RNA was reverse transcribed into cDNA for 2 h at 42°C. cDNA (0.1 µg) was subjected to qPCR using an iQ SYBR-Green system (Bio-Rad Laboratories, Inc.). All primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) and the sequences used were as follows: PI3K forward, 5′-CTCATGCCAGGCACTGTGCTA-3′ and reverse, 5′-GAATCAAGGCACACCTGTGGAA-3′; TIPE-2 forward, 5′-GGAACATCCAAGGCAAGACTG-3′ and reverse, 5′-AGCACCTCACTGCTTGTCTCATC-3′; and β-actin forward, 5′-GACTACCTCATGAAGATCCTCACC-3′ and reverse, 5′-TCTCCTTAATGTCACGCACGATT-3′. PCR was performed with the following thermocycling conditions: 45 amplification cycles of 96°C for 30 sec, annealing at 63°C for 20 sec, with touchdown to 54°C for 20 sec and extension at 72°C for 5 min. Results were expressed as a fold change by comparing levels of target mRNA expression to that of the reference gene using the 2^−ΔΔCq method (25).

HepG2 cells were treated with TIPE-2 (2.0 mg/ml) for 24 h and were then homogenized with lysis buffer containing protease-inhibitor (cat. no. P3480; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Protein concentration was measured with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.), protein (10 µg/lane) was separated by 12.5% SDS-PAGE and subsequently transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% milk for 2 h at 37°C prior to incubation with the following primary antibodies overnight at 4°C: PI3K (1:1,000; cat. no. ab191606), AKT (1:1,000; cat. no. ab8805), neuroblastoma Ras viral oncogene (N-ras; 1:1,000; cat. no. ab206969), P27 (1:1,000; cat. no. ab191606), phosphorylated (p)AKT (1:1,000; cat. no. ab81283), binding immunoglobulin protein (BIP; 1:1,000; cat. no. ab108615), P53 (1:1,000; cat. no. ab1431), B-cell lymphoma 2 (Bcl-2; 1:1,000; cat. no. ab59348), cyclin D1 (1:1,000; cat. no. ab134175), cyclin-dependent kinase (CDK)1 (1:1,000; cat. no. ab131450), CDK2 (1:1,000; cat. no. ab32147), vimentin (VIM; 1:1,000; cat. no. ab92547), collagen type I (CT-I; 1:1,000; cat. no. ab34710), Slug (1:1,000; cat. no. ab27568), c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. ab124956), nuclear factor (NF)-κB (1:1,000; cat. no. ab28849), nuclear factor (erythroid-derived 2)-like 2 (NRF2) (1:1,000; cat. no. ab62352), caspase-3 (1:1,000; cat. no. ab13847), caspase-8 (1:1,000; cat. no. ab25901), glucose-regulated protein 78 (GRP78; 1:1,000; cat. no. ab21685), CCAAT-enhancer-binding protein homologous protein (CHOP; 1:1,000; cat. no. ab179823), eukaryotic initiation factor 2 (eIF2α; 1:1,000; cat. no. ab32713), peIF2α (1:1,000; cat. no. ab214434) and β-actin (1:1,000; cat. no. ab827; Abcam, Shanghai, China) for 12 h at 4°C. Membranes were then incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated immunoglobulin G secondary antibody (1:2,000; cat. no. PV-6001; OriGene Technologies, Inc., Beijing, China) for 24 h at 4°C. The blots were visualized using a chemiluminescence detection system (GE Healthcare, Chicago, IL, USA). Relative protein expression levels were calculated using Quantity-One software (version 3.0; Bio-Rad Laboratories, Inc.) and are presented as n-fold of β-actin expression levels.

HepG2 cells were treated with TIPE-2 (2.0 mg/ml) for 24 h; untreated cells were used as a control. Migration and invasion assays of HepG2 cells were conducted in 6-well culture plates with chamber inserts (BD Biosciences, San Diego, CA, USA). For migration assays, 1×10⁶/well HepG2 cells were placed into the upper chamber with DMEM medium. The lower chamber contained DMEM medium with 0.1% FBS. For invasion assays, cells (1×10⁶/well) were placed into the upper chamber with a Matrigel-coated insert. After 24 h culture at 37°C, migratory and invasive cells were fixed with 1.4% paraformaldehyde for 30 min at 37°C and were stained for 30 min in 0.1% crystal violet solution in PBS. Migration and invasion of HepG2 cells was counted in at least three randomly selected fields per membrane using a light microscope (Olympus BX51; Olympus Corporation, Tokyo, Japan).

HepG2 cells were cultured until they reached 85% confluence. Apoptotic rate was assessed following incubation with TIPE-2 (2.0 mg/ml) for 48 h. Briefly, HepG2 cells were trypsinized and collected, the cells were then washed in cold PBS, adjusted to 1×10⁸ cells/ml with PBS, and labeled with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; Annexin V-FITC kit; BD Biosciences) for 2 h at 4°C. Apoptotic rate was analyzed using FCS Express^™ IVD software (version 4; De Novo Software, Glendale, CA, USA).

HepG2 cells were cultured in 6-well plates until they reached 85% confluence and the medium was then removed. Subsequently, HepG2 cells were washed three times and were incubated with DMEM supplemented with 10% FBS and TIPE-2 (2.0 mg/ml) for 24 h. Subsequently, the supernatant was removed and cells were incubated with Triton X-100 (1%) for 30 min. Lactate dehydrogenase activity in cell lysates was used to analyze cell viability using the Promega CytoTox 96 assay kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol.

To analyze the effects of TIPE-2 (2 mg/ml) on the HepG2 cell cycle, flow cytometry was performed. HepG2 cells in the exponential phase of growth were treated with TIPE-2 (2 mg/ml) for 48 h at 37°C. Cells were subsequently washed with PBS, trypsinized (1% trypsin) for 5 min at 37°C and rinsed with PBS. Cells were fixed in 75% ice-cold ethanol for 15 min and washed with PBS three times. RNaseA (20 µg ml/l; Fermentas; Thermo Fisher Scientific, Inc.) was added to fixed cells, which were subsequently stained with PI (20 µg ml/l; Sigma-Aldrich; Merck KGaA) for 10 min at 37°C. The percentage of cells in the G1, G2 and S phase were analyzed using a cell cycle analysis kit (cat. no. C34568; Invitrogen; Thermo Fisher Scientific, Inc.), a BD FACSCalibur flow cytometer (BD Biosciences) and FCS Express IVD software (version 4; De Novo Software).

A total of 80 specific pathogen-free (SPF) female BALB/c nude mice (age, 6–8 weeks) were purchased from Harbin Veterinary Research Institute (Harbin, China). All mice were feed under pathogen-free conditions. Mice were maintained at 22–24°C in a 12 h light/dark cycle with ad libitum access to food and water. A total of 5×10⁷ HepG2 cells were injected into the right flank of female BALB/c nude mice at a total volume of 200 µl. Tumor-bearing mice then underwent intratumoral injection with TIPE-2 (6.0 mg/ml) or PBS (n=40/group), once tumor diameters reached 5–8 mm on day 6 after tumor inoculation. The treatment was continued 15 times at intervals of every two days for a total of 30 days. Tumor diameters were recorded once every 2 days and tumor volume was calculated using the following formula: 0.52 × smallest diameter² × largest diameter. Survival analysis was conducted over 120 days to analyze the therapeutic effects of TIPE-2 in tumor-bearing mice.

Immunohistochemical staining was performed according to the avidin-biotin-peroxidase technique. HCC tissues were isolated from experimental mice and paraffin-embedded tissue sections (4 µm) were prepared and epitope retrieval was performed by heating the tissue sections at 100°C for 30 min in a citrate solution (10 mmol/l; pH 6.0) followed by dewaxing in xylene and rehydrating in a graded ethanol series for further analysis. Subsequently, paraffin-embedded sections were treated with hydrogen peroxide (3%) for 10–15 min and were blocked in 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 10–15 min at 37°C. Finally, the sections were incubated with biotinylated goat anti-mouse caspase-3 (1:1,000; cat. no. ab13847), caspase-9 (1:1,000; cat. no. ab32539), PI3K (1:1,000; cat. no. ab191606), AKT (1:1,000; cat. no. ab8805), GRP78 (1:1,000; cat. no. ab21685) and CHOP (1:1,000; cat. no. ab179823) antibodies (Abcam) at 4°C for 12 h. Samples were washed three times with PBS and then incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:2,000, cat. no. PV-6001; OriGene Technologies, Inc.) for 2 h at 37°C. 3,3′-diaminobenzidene (0.05%) was used as the chromogen for 30 min at 37°C and 1% hematoxylin as the nuclear counterstain for 30 min at 37°C. The relative protein expression levels were analyzed using a chemiluminescence detection system (GE Healthcare). Tumor tissue images were captured with a ZEISS LSM 510 confocal microscope (magnification, ×40; Zeiss AG, Oberkochen, Germany). Relative protein expression levels were determined using Quantity-One software 3.0 (Bio-Rad Laboratories, Inc.) and are presented as the n-fold of β-actin expression levels.

HepG2 cells were treated with TIPE-2 (2 mg/ml) for 12 h at 37°C. Following this, cells were washed with PBS at room temperature and fixed with 4% paraformaldehyde for 1 h at 37°C. The cells were washed again with PBS three times, blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 2 h at 37°C and subsequently stained with the following antibodies for 12 h at 4°C: Ki67 (1:1,000; cat. no. ab15580; Abcam), PCNA (1:1,000; cat. no. ab18197; Abcam), E-cadherin (1:1,000; cat. no. ab40772; Abcam) and fibronectin (1:1,000; cat. no. ab2413; Abcam). Cells were washed with PBS and subsequently incubated with an Oregon Green^® 488-conjugated IgG (1:1,000; cat. no. O-6382; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h in a light-protected chamber at 37°C. Immunofluorescence signals were detected using a laser scanning confocal microscope (magnification, ×40; Zeiss AG).

All data are presented as the mean ± standard error of the mean of triplicate experiments. SPSS 19.0 software (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Unpaired data were analyzed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1A and B, TIPE-2 administration (0.5, 1, 1.5, 2.0 and 2.5 mg/ml) significantly inhibited HepG2 cell growth in a dose- and time-dependent manner. The results indicated that 2.0 mg/ml TIPE-2 markedly suppressed HCC cell proliferation and increased the number of cells in S phase of the cell cycle (Fig. 1C and D). Western blot analysis demonstrated that TIPE-2 administration (2.0 mg/ml) decreased the expression levels of cyclin D1, CDK1 and CDK2 in HepG2 cells (Fig. 1E). Immunofluorescence indicated that TIPE-2 administration (2.0 mg/ml) decreased the expression levels of Ki67 and proliferating cell nuclear antigen in HepG2 cells (Fig. 1F). These results suggested that TIPE-2 may significantly inhibit HCC cell growth and proliferation in vitro.

Local migration and long-distance invasion are the most common characteristics of HCC. The present study investigated migration and invasion of HepG2 cells following treatment with TIPE-2 (2.0 mg/ml). Migration and invasion assays demonstrated that TIPE-2 significantly suppressed HepG2 cell migration and invasion in vitro (Fig. 2A and B). Western blot analysis demonstrated that TIPE-2 treatment decreased the expression levels of VIM, CT-I and Slug in HepG2 cells (Fig. 2C). In addition, TIPE-2 decreased JNK and NF-κB expression in HepG2 cells (Fig. 2D). Immunofluorescence demonstrated that E-cadherin/DNA and fibronectin/DNA expression levels were decreased in HepG2 cells following TIPE-2 treatment (Fig. 2E). HepG2 cell viability was also decreased by TIPE-2 treatment (Fig. 2F). These results indicated that TIPE-2 may significantly inhibit migration and invasion of HepG2 cells via inhibiting the expression levels of cancer cell migration-associated proteins.

As shown in Fig. 3A, TIPE-2 administration (2.0 mg/ml) promoted apoptosis of HepG2 cells after 48 h. Western blotting demonstrated that the expression levels of caspase-3 and caspase-8 were increased by TIPE-2 administration (Fig. 3B). In addition, TIPE-2 administration downregulated P53 and Bcl-2 expression levels in HepG2 cells (Fig. 3C). Results also indicated that the expression and levels of eIF2α and peIF2α were upregulated in HepG2 cells following TIPE-2 treatment (Fig. 3D). Furthermore, the expression levels of NRF2 and BIP were increased in HepG2 cells following TIPE-2 treatment (Fig. 3E). The expression levels of GRP78 and CHOP were also increased in HepG2 cells following TIPE-2 treatment (Fig. 3F). These results suggested that TIPE-2 may promote HCC apoptosis in vitro.

As shown in Fig. 4A, the present study demonstrated that TIPE-2 significantly inhibited PI3K and AKT expression in HepG2 cells. In addition, N-ras and P27 expression were decreased by TIPE-2 treatment in HepG2 cells (Fig. 4B). pAKT levels were also decreased by TIPE-2 treatment in HepG2 cells when compared with pAKT levels in the control group (Fig. 4C). We observed that TIPE-2 decreased ratio of pAkt:Akt in HepG2 cells (Fig. 4D). As shown in Fig. 4E and F, pedue12.4-PI3K or pedue12.4-TIPE-2 upregulated PI3K and TIPE-2 mRNA expression, respectively, compared with pedue12.4-vector in HepG2 cells. Mechanistic analysis indicated that PI3K overexpression alone (PI3KOR) inhibited the apoptosis of HepG2 cells, whereas TIPE-2 treatment abolished this effect (Fig. 4G). In addition, N-ras and BIP expression was increased by PI3KOR in HepG2 cells (Fig. 4H). In addition, PI3KOR increased the expression levels of P53 and Bcl-2 in HepG2 cells when compared to the control group (Fig. 4I). These results suggested that TIPE-2 may regulate HCC cell apoptosis through regulation of the PI3K/AKT signaling pathway.

The present study further investigated the antitumor effects of TIPE-2 treatment on HepG2-bearing nude mice. In order to avoid the interference of pathogenic microorganisms, SPF female BALB/c mice (body weight, 30–35 g) were implanted with HepG2 cells and treated with TIPE-2. As shown in Fig. 5A, TIPE-2 treatment (6 mg/kg) inhibited tumor growth during the 30-day observation period compared with in the control group. TUNEL assay demonstrated that TIPE-2 treatment increased the number of apoptotic cells in tumor tissues compared with in the PBS group (Fig. 5B). In addition, TIPE-2 treatment increased the expression levels of caspase-3 and caspase-8 (Fig. 5C), and upregulated PI3K and AKT in tumor tissues (Fig. 5D). Furthermore, TIPE-2 treatment increased GRP78 and CHOP expression in tumor tissues (Fig. 5E). Notably, survival of HepG2-bearing mice was prolonged by TIPE-2 treatment during the 120-day experiment (Fig. 5F). These results suggested that TIPE-2 may be a potential anticancer agent for the treatment of HCC.