The human normal hepatocyte cell line HHL-5 and the HCC cell line MHCC97-H were obtained from the American Type Culture Collection. Both cell lines were cultured in DMEM (Sigma-Aldrich; Merck KGaA) supplemented with 10% fetal bovine serum (FBS; Wisent Biotechnology) and 1% penicillin-streptomycin (Sigma-Aldrich; Merck KGaA) in an incubator with 5% CO₂ at 37°C. For OP-B treatment, cells were cultured in DMEM containing different concentrations (5, 10, 20 and 40 µM) of OP-B (purity >97%; Shanghai Yuanye Bio-Technology Co., Ltd.) for 24 h as previously described (8,11,17,18). Cells cultured in normal DMEM without OP-B were used as a control.

A PTP1B overexpression vector (Ov-PTP1B) was constructed by cloning human PTP1B cDNA into the pcDNA3.1 vector (Thermo Fisher Scientific, Inc.). Empty pcDNA3.1 vector was used as negative control (Ov-NC). MHCC97-H cells were grown in six-well plates and subsequently transfected with 2 µg of either vector using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 24 h according to the manufacturer's protocol. At 48 h post-transfection, monoclonal cells were then selected and examined for PTP1B overexpression.

A total of 15 male BALB/c nude mice (age, 4–5 weeks; 15–20 g; Shanghai Laboratory Animal Center) were maintained under specific pathogen-free conditions at room temperature under a controlled 12/12 h light/dark cycle, and received food and water ad libitum. The animal study protocol was approved by the Ethics Committee of Suzhou Hospital of Integrated Traditional Chinese and Western Medicine (Suzhou, China; approval no. 20180901). To establish the HCC xenograft model, MHCC97-H cells (2×10⁶ cells/200 µl) were subcutaneously injected into the right flank of the nude mice. After six days, mice were treated by oral gavage with normal saline (control group; n=5), 15 mg/kg OP-B (OP-B 15 mg/kg group; n=5) or 75 mg/kg OP-B (OP-B 75 mg/kg group; n=5) once a day for 21 days, as previously described (8,17,18). Mouse body weight and tumor volume were recorded every 7 days; at 21 days post-treatment, the mice were sacrificed by cervical dislocation under anesthesia with 1% sodium pentobarbital (50 mg/kg intraperitoneally). Tumor volumes for each mouse were calculated as follows: Tumor volume=1/2 × length × width².

Xenografted tumor tissues were fixed with 4% paraformaldehyde at 4°C for 12 h and embedded in paraffin. After being deparaffinized with xylene and rehydrated with a descending ethanol series, slides (3 µm thickness) were stained with the IHC Assay Kit (cat. no. KGOS300; Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol and then incubated at 4°C overnight with the following primary antibodies purchased from Abcam: Anti-Ki67 (1:1,000; cat. no. ab15580), anti-CD31 (1:50; cat. no. ab28364) and anti-VEGFA (1:200; cat. no. ab52917). Secondary antibody goat anti-rabbit IgG (1:1,000; cat. no. ab6721; Abcam) was applied at room temperature for 2 h. Images of the tissue sections were then captured with a fluorescence microscope (DMi8; Leica Microsystems GmbH; ×200 magnification).

Cell Counting Kit-8 (CCK8; Beyotime Institute of Biotechnology) was used to measure cell viability. Briefly, cells were seeded into 96-well plates at a density of 1,000 cells/well at 37°C for 24 h. After treatment with different concentrations of OP-B (0, 5, 10, 20 and 40 µM) at 37°C for 24 h, cells were incubated with 10 µl of CCK-8 solution at 37°C for 2 h. The optical density was calculated at 450 nm using a microplate reader (Model550; Bio-Rad Laboratories, Inc.).

For the colony formation assay, 1×10³ MHCC97-H cells were cultivated in six-well plates in a 37°C incubator with 5% CO₂. After 14 days, cell colonies were fixed with 95% alcohol at room temperature for 30 min and stained with 0.1% crystal violet at room temperature for 30 min (Sigma-Aldrich; Merck KGaA). Images were captured with a light microscope (Nikon Corporation; ×200 magnification) and colonies were counted using ImageJ software (version 1.8.0; National Institutes of Health).

According to the manufacturer's protocol, MHCC97-H cell apoptosis was analyzed using TUNEL Assay kit (Abcam). Briefly, cells were fixed in 4% paraformaldehyde solution for 30 min at room temperature, treated with 0.2% Triton X-100 for 5 min at room temperature, washed twice in PBS at room temperature, and labeled with fluorescein-12-dUTP using terminal deoxynucleotidyl transferase for 2 h at room temperature. Subsequently, cell nuclei were stained with 5 µg/ml DAPI at room temperature for 3 min. All images were obtained using a fluorescence microscope (DMi8; Leica Microsystems GmbH; ×200 magnification).

Briefly, MHCC97-H cells were seeded in a six-well plate at a density of 5×10⁵ cells/well in DMEM. When the cells were ~80% confluent, scratches were made in the middle of slides using a sterile 10-µl pipette tip. After incubation for another 48 h with or without 20 µM OP-B in serum-free DMEM at 37°C, images were captured to estimate closure of the gap using a light microscope (Nikon Corporation; ×200 magnification). Migration distance was evaluated using ImageJ software (version 1.8.0; National Institutes of Health) and calculated as follows: Migration distance=(width of gap at 0 h-width of gap at 48 h)/width of gap at 0 h. Data are shown as relative migration distance by normalization to the control group.

The invasive ability of MHCC97-H cells was evaluated by the Biocoat invasion assay kit (Corning, Inc.) strictly following the manufacturer's protocol. Firstly, 5×10⁵ cells per well were plated in the upper chamber followed by treatment with 20 µM OP-B or not. After 12 h of treatment, the cells in the upper chamber were incubated with serum-free DMEM medium, whereas media supplemented with 10% FBS was placed at the lower chamber. After 48 h, the invasive cells at the lower chamber were stained with 0.1% crystal violet at room temperature and observed using an optical light microscope (Leica Microsystems GmbH; ×200 magnification).

Matrigel (BD Biosciences) was thawed at 4°C, pipetted into 96-well plates and allowed to polymerize at 37°C for 1 h. Cells (1×10⁴ per well) were suspended in DMEM containing 0 or 20 µM OP-B and seeded onto the Matrigel. After incubation for 24 h at 37°C, tube formation was analyzed by counting nodes and measuring total tube numbers using ImageJ software (version 1.8.0; National Institutes of Health).

Total RNA was extracted using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, and then reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. SYBR-Green Supermix (Takara Biotechnology Co., Ltd.) was used for qPCR in a 10 µl reaction volume on the Roche Light Cycler R480 System (Roche Diagnostics). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 30 sec; followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec and extension at 70°C for 10 sec. Target gene expression levels were analyzed using the 2^−ΔΔCq (19) method and normalized to GAPDH. The primers used for RT-qPCR were as follows: PTP1B forward, 5′-CCAGCCAAAGGGGAGCCGTC-3′ and reverse, 5′-CTATGTGTTGCTGTTGAACA-3′; and GAPDH forward, 5′-AATGGGCAGCCGTTAGGAAA-3′ and reverse, 5′-GCGCCCAATACGACCAAATC-3′.

Total protein was extracted from cells or tumor tissues by RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a BCA Kit (Beyotime Institute of Biotechnology), mixed with 5X sample buffer and boiled at 95°C for 5 min. Equal amounts (40 µg) of protein lysate per sample were separated on 10% gels using SDS-PAGE and transferred onto the PVDF membranes. After blocking with 5% skimmed milk for 2 h at room temperature, membranes were incubated with primary antibodies at 4°C overnight and secondary antibodies (goat anti-rabbit IgG HRP; 1:10,000; cat. no. ab6721; Abcam) at room temperature for 2 h. An enhanced chemiluminescence (ECL) detection kit (Amersham; Cytiva) was used to visualize the protein bands. Band intensity was semi-quantified by ImageJ software (v1.8.0; National Institutes of Health). Primary antibodies (all from Abcam) included: anti-PTP1B (1:1,000; cat. no. ab252928), anti-Bcl-2 (1:1,000; cat. no. ab32124), anti-Bax (1:5,000; cat. no. ab32503), anti-cleaved caspase 3 (1:500; cat. no. ab32042), anti-caspase-3 (1:1,000; cat. no. ab184787), anti-cleaved poly ADP-ribose polymerase (PARP; 1:5,000; cat. no. ab32064), anti-PARP (1:5,000; cat. no. ab191217), anti-E-cadherin (1:10,000; cat. no. ab40772), anti-N-cadherin (1:10,000; cat. no. ab76011), anti-Vimentin (1:1,000; cat. no. ab45939), anti-VEGFA (1:10,000; cat. no. ab52917), anti-phosphorylated (p)-phosphatidylinositol 3 kinase (PI3K; 1:1,000; cat. no. ab191606), anti-total (t)-PI3K (1:1,000; cat. no. ab278545), anti-p-AKT (1:1,000; cat. no. ab38449), anti-t-AKT (1:1,000; cat. no. ab8805), anti-p-adenosine 5′-monophosphate-activated protein kinase (AMPK; 1:1,000; cat. no. ab109402), anti-t-AMPK (1:1,000; cat. no. ab214425) and anti-GAPDH (1:10,000; cat. no. ab181602).

Data are expressed as the mean ± standard deviation. One-way ANOVA followed by Tukey's post hoc test was used for analysis between multiple groups. Calculations were performed using GraphPad Prism software (version 5.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

The anticancer effects of OP-B on HCC were first investigated in vivo. HCC xenografts were injected into mice (Fig. 1A) that were subsequently treated orally with 0, 15 or 75 mg/kg OP-B. Tumor volume and mouse body weight were examined for 21 days (Fig. 1B). The tumors were excised (Fig. 1C) and the final tumor weight and the tumor/body weight ratio were measured. (Fig. 1D), which were significantly reduced in mice treated with both 15 and 75 mg/kg OP-B compared with the control mice. Additionally, it was observed from IHC results that the expression level of Ki67 (Fig. 1E), CD31 (Fig. 1F) and VEGFA (Fig. 1G) in HCC tumor tissues of the xenografted mice was markedly decreased upon OP-B (15 and 75 mg/kg) administration compared with the control mice. In addition, the mRNA and protein expression levels of PTP1B in tumor samples of xenograft mice model was significantly inhibited by OP-B (Fig. 1H and I, respectively). These results showed that OP-B exerted anticancer effect on HCC in vivo.

To validate the effect of OP-B on HCC and PTP1B expression in vitro, HHL-5 human normal hepatocyte and MHCC97-H HCC cells were exposed to different concentrations of OP-B (5, 10, 20 and 40 µM) for 24 h, then cell viability was measured. As revealed in Fig. 2A, OP-B treatment did not cause significant effect on HHL-5 cell viability, but significantly reduced MHCC97-H cell viability in a concentration-dependent manner. In addition, both the mRNA and the protein expression levels of PTP1B were significantly downregulated by OP-B (Fig. 2B and C, respectively).

Subsequently, PTP1B was overexpressed in MHCC97-H cells by transfection of Ov-PTP1B vector and then the overexpression efficiency was confirmed by RT-qPCR and western blotting (Fig. 2D and E, respectively).

To observe the effect of OP-B on the malignant processes of HCC, MHCC97-H cells overexpressing PTP1B were treated with 20 µM OP-B, then cell proliferation, apoptosis, migration, invasion and angiogenesis were evaluated. As revealed in Fig. 3A, OP-B significantly reduced cell viability compared with the untreated control group, whereas OP-B + Ov-PTP1B treatment significantly enhanced the cell viability compared with the OP-B + Ov-NC group. These results indicated that the inhibitory effect of OP-B on HCC cell viability was reversed by PTP1B overexpression. Furthermore, OP-B markedly suppressed the colony formation compared with control MHCC97-H cells, whereas PTP1B overexpression markedly reduced this effect (Fig. 3B). TUNEL staining revealed that apoptosis was significantly increased upon OP-B treatment compared with the control group, and PTP1B overexpression reduced the level of apoptosis compared with OP-B + Ov-NC (Fig. 3C). Consistently, OP-B treatment resulted in decreased protein expression of Bcl-2, but increased expression levels of Bax, cleaved caspase 3 and cleaved PARP, suggesting the induction of cell apoptosis (Fig. 3D). However, PTP1B overexpression caused a reverse effect on the expression of these proteins in OP-B-treated cells (Fig. 3D).

Results from wound healing, invasion and tube formation assays demonstrated that OP-B significantly inhibited MHCC97-H cell migration, invasion and angiogenesis compared with untreated control cells (Fig. 4A-C, respectively). By contrast, OP-B + Ov-PTP1B treatment significantly increased these processes compared with OP-B + Ov-NC group (Fig. 4A-C). In addition, OP-B treatment significantly increased E-cadherin expression but decreased N-cadherin, Vimentin and VEGFA expression (Fig. 4D), whereas PTP1B overexpression reversed the effect on the expression levels of these proteins caused by OP-B.

To uncover the underlying related signaling mechanism of OP-B, the expression of the PI3K/AKT and AMPK signaling pathways was detected. As demonstrated in Fig. 5, MHCC97-H cells showed decreased p/t-PI3K and p/t-AKT expression levels as well as increased p/t-AMPK expression in response to OP-B treatment. Conversely, when compared with OP-B + Ov-NC, OP-B + Ov-PTP1B treatment significantly upregulated p/t-PI3K and p/t-AKT expression levels and downregulated p/t-AMPK expression. These data indicated that OP-B may inhibit PI3K/AKT activation and activate the AMPK signaling, which is supported by the data showing that these effects were blocked by PTP1B overexpression.