GYY4137 was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). STAT3, p-STAT3 (Y705), p-STAT3 (S727), JAK, p-JAK2, Bcl-2, Mcl-1, cyclin D1, survivin, HIF-1α, VEGF, cleaved-poly(ADP-ribose) polymerase (PARP), cleaved-caspase-9 and cleaved-caspase-3 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). β-actin antibody was purchased from Sigma (St. Louis, MO, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), RNase and sodium-orthovanadate and propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

HCC cell lines HepG2, and Bel7402, and hepatocellular LO2 cells were obtained from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). All cells were cultured in a humidified atmosphere with a 5% CO₂ incubator at 37°C.

HepG2, Bel7402 and LO2 cells were incubated in triplicate in a 96-well plate at a density of 1×10⁴ cells with 100 μl culture medium per well in the presence or absence of indicated concentrations of GYY4137 for 24, 48 and 72 h. After which, cell viability was determined by the MTT dye uptake method as described earlier (20).

HepG2 and Bel7402 cells were cultured in 6-well plates at a density of 5×10⁵ cells/ml in a CO₂ incubator overnight and treated with GYY4137. The total protein was prepared as previously described (21). The equalized amounts of proteins from each sample were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 1% (w/v) bovine serum albumin (BSA) for 2 h, and then incubated with the primary antibody overnight at 4°C. Primary antibodies were STAT3, p-STAT3 (Y705), p-STAT3 (S727), JAK2, p-JAK2 (Y1007/1008), Bcl-2, Mcl-1, cyclin D1, survivin, HIF-1α, VEGF, cleaved-PARP, cleaved-caspase-9, cleaved-caspase-3 (Cell Signaling Technology) and β-actin (Sigma). Then the membranes were incubated with secondary antibody conjugated with IgG horseradish peroxidase (HRP) for 1 h at room temperature and immune complexes were detected by the enhanced chemiluminescence system. β-actin served as a loading control.

HepG2 and Bel7402 cells were incubated in 6-well plates in the presence of different concentrations of GYY4137 for 24 h. Thereafter, treated cells were fixed and incubated with RNase and propidium iodide (PI) in PBS. Cell cycle distribution was analyzed with a FACScan laser flow cytometer (FACSCalibur, Becton-Dickinson, Franklin Lakes, NJ, USA). The data were analyzed using the software CELL Quest.

Apoptosis was measured with caspase-3/7 assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Briefly, cells were seeded into a 96-well plate. After the treatment, 100 ml of Apo-One Caspase-3/7 reagent was added to each well and was incubated at 37°C for 30 min. The fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

For the measurement of VEGF, 5×10⁵ U266 cells/well were seeded in 12-well plates and grown to 75–80% confluence, then cells were switched to fresh serum-free mediumin the presence or absence of rosiglitazone and ATRA and incubated for another 12 h. Cell-free culture supernatants were harvested and assayed for secreted VEGF using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA).

Male BALB/c-nu/nu mice (4–6 weeks old, Slac Laboratory Animal, Shanghai, China) were kept in the animal facilities at the Nanjing Medical University and maintained under specific pathogen-free conditions. All animal procedures were conducted according to the guidelines approved by the China Association of Laboratory Animal Care. Cultured HepG2 cells (5×10⁶) suspended in 0.2 ml PBS were injected into the right flank of mice. The mice were kept in a pathogen-free environment, 5–7 days later when the tumor volume reached 100 mm³, the mice were divided into four groups (control group and three-dose GYY4137 groups) in a manner to equalize the mean tumor among the four groups (n=7 each). GYY4137 at 50, 20 and 10 mg/kg dose suspended in 0.5% carboxymethyl cellulose (CMC) was given as gavage to mice daily for 4 weeks, and mice of control group were given 0.1 ml 0.5% CMC solution. The tumor size was measured in two orthogonal directions using calipers every three days, and the tumor volume (mm³) was estimated using the equation length × (width)² × 0.5. Four weeks later, the mice were sacrificed and the tumors were resected. Part of tumor tissues were homogenized and subjected to western blot assay. The primary antibodies were STAT3, p-STAT3 (Y705), p-STAT3 (S727), Bcl-2, Mcl-1, cyclin D1, survivin, HIF-1α, VEGF (Cell Signaling Technology) and β-actin (Sigma).

Immunohistology analysis was carried out using paraffin section. Paraffin section were incubated in a blocking solution (10% donkey serum + 5% non-fat dry milk + 4% BSA + 0.1% Triton X-100) for 10 min and then hydrated sections were incubated at 4°C overnight with anti-p-STAT3 (Y705) and p-STAT3 (S727) or anti-STAT3 antibody, respectively. After washing with PBS, the sections were incubated with diluted (1:200) biotinylated secondary antibody for 30 min. Subsequently, the slides were washed again in PBS and incubated for 30 min with the preformed avidin-horseradish peroxidase macromolecular complex. Development of peroxidase reaction was achieved by incubation in 0.01% 3,3-diaminobenzidine tetrahydrochloride (DAB) in PBS containing 0.01% hydrogen peroxide for approximately 5 min at room temperature. Sections were then washed thoroughly in tap water, counterstained in haematoxylin, dehydrated in absolute alcohol, cleared in xylene and mounted in synthetic resin for microscopic examination.

Statistical difference was analyzed by two-way Student’s t-test. P<0.05 was considered to indicate statistical significance. The values are expressed as the mean ± SD. Three or more separates experiments were performed.

To examine whether the antitumor activity of GYY4137 may be due to the inhibition of IL-6/STAT3 pathway, we evaluated the effects of GYY4137 on IL-6-induced STAT3 phosphorylation in human HCC cells. HepG2 and Bel7402 cell lines with higher IL-6 and phosphorylated STAT3 were treated with GYY4137 for 24 h. As shown in Fig. 1A, IL-6 significantly activated STAT3 phosphorylation at Y705 both in HepG2 and Bel7402 cells. GYY4137 inhibited the IL-6-induced p-STAT3 (Y705) in a dose-dependent manner, whereas it had no effect on p-STAT3 (S727) and total STAT3 (Fig. 1A). Furthermore, inhibition was evident as early as 4 h after treatment and lasted for 24 h (Fig. 1B) in HCC cell lines. These findings indicated that GYY4137 inhibited IL-6-induced STAT3 activation in human HCC cells.

JAK2 is considered as one of the most common activators of STAT3. To explore the mechanism of the inhibitory effects on p-STAT3 (Y705), the levels of JAK2 phosphorylation were evaluated. P-JAK2 (Y1007/1008) was suppressed in HepG2 and Bel7402 cell lines with GYY4137 treatment, whereas basic JAK2 was not detectable (Fig. 2A). To further investigate whether GYY4137 would affect STAT3 downstream genes, we used western blot analysis to examine the expression of Bcl-2, Mcl-1, cyclin D1 and survivin. As shown in Fig. 2B, GYY4137 treatment downregulated the expression of these proteins both in HepG2 and Bel7402 cell lines.

To examine the growth inhibitory effect of GYY4137, we first assessed cell cycle distribution by flow cytometric analysis. As shown in Fig. 3, GYY4137 increased the number of cells in the G0/G1 phase in HepG2 and Bel7402 cells. The results are consistent with the inhibitory effect of GYY4137 on cyclin D1 in these cells.

Since IL-6/STAT3 targets certain genes such as Bcl-2, Mcl-1 and survivin involved in anti-apoptosis, we next examined the effect of GYY4137 on apoptosis induction. As shown in Fig. 4A, GYY4137 promoted the cleavage of proapoptotic 116 kDa full-length PARP in an 89 kDa fragment, a process known to impair genomic integrity before apoptosis. We also observed that the levels of cleaved caspase-9 and caspase-3 were increased by treatment with GYY4137 in a dose-dependent manner (Fig. 4A). Flow cytometric data revealed that treatment with 200 and 400 μM of GYY4137 increased the number of cells in the subG1 phase (13–20%), indicating that the HCC cells were undergoing apoptosis (Fig. 4B). MTT assay showed that GYY4137 inhibited tumor cell viability time- and dose-dependently (Fig. 4C), while it showed weak inhibition on normal hepatocellular LO2 cells only after 400 μM GYY4137 treatment for 72 h (data not shown). These results showed that GYY4137 was able to induce apoptosis in HCC cells through regulating the caspase pathway.

A low oxygen level is a characteristic feature of solid tumors and a negative prognostic factor for cancer patient survival. The response of cancer cells to hypoxia not only drives neo-angiogenesis, but also enhances cancer cell survival and malignant phenotype development. Hypoxia is known to increase the expression of HIF-1α, a major regulator of pro-angiogenic signaling. Since HIF-1α is the major transcriptional modulator of angiogenic factors such as VEGF, we evaluated the effect of GYY4137 on hypoxia induced expression of HIF-1α and VEGF in HCC cells. The cells were treated with various concentrations of GYY4137 under hypoxia mimicking condition induced by 100 μM CoCl₂ for 6 h. As shown in Fig. 5A, GYY4137 suppressed the expression of HIF-1α, which was increased after exposure to hypoxia. To further confirm the effect of GYY4137 on hypoxia-induced VEGF, an immediate downstream target gene of HIF-1α, VEGF protein level secreted to media was measured by ELISA in HepG2 cells. A notable increase of VEGF was observed under the hypoxic condition, whereas the treatment of GYY4137 suppressed VEGF production dose-dependently (Fig. 5B).

To determine the antitumor activity of GYY4137 in vivo, we evaluated its effect in a nude mouse xenograft model of HepG2 cells. GYY4137 was administered for 4 weeks. After 4 weeks, the mice were sacrificed and the tumor mass was dissected. The high dose of GYY4137 (50 mg/kg/d) inhibited tumor growth significantly (P<0.05) after administration for 13 days, and the low dose (10 mg/kg) of GYY4137 exhibited no significant inhibitory effect on tumor growth (Fig. 6A). Body weight of the mice was observed during administration of GYY4137. There was no significant weight loss in mice treated with GYY4137 of middle and high dose groups, while significant weight loss was detected in mice treated with GYY4137 of low dose or saline groups (Fig. 6B).

To confirm whether the inhibition of GYY4137 on xenograft tumor growth was mediated by blocking STAT3 signaling, we investigated the expression levels of p-STAT3 and STAT3 in xenograft tumor of mice treated with GYY4137 by IHC analysis. As shown in Fig. 7A and B, the level of p-STAT3 (Y705) was decreased in xenograft tumors of mice treated with GYY4137 compared with the model xenograft tumors, whereas the level of STAT3 had no significant change in the model group or the GYY4137 treated group. In concert with the decreased expression of p-STAT3, a clear downregulation in the target gene expression of STAT3, such as cyclin D1, Mcl-1, HIF-1α and VEGF, were found in the xenograft tumor tissue of the GYY4137-treated mice, in a GYY4137 dose-dependent manner (Fig. 7C). Results in vivo further confirmed that the antitumor effect of GYY4137 was partly mediated by blocking the STAT3 signaling pathway.