AC was purchased from Kyung Hee Herb Pharm (Wonju, Korea), a licensed herb company. AC was pulverized and extracted with 70% EtOH under reflux three times, and the extract was filtered and evaporated on a rotary evaporator under reduced pressure and then lyophilized. ACE was subjected to LC-MS/MS analysis and Scopoletin and liquiritin were found to be present at concentrations of 393.3±45.7 µg/g and 39.33±6.3 µg/g, respectively.

Huh7 and HepG2 human hepatocellular carcinoma cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 U/ml). NIH3T3-W4P cell line expressing W4P mutant LHB was established and maintained as previously described (4).

Anti-proliferative effects of ACE on HCC cells were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Huh7 and HepG2 cells were treated with increasing concentrations of ACE for 72 h followed by further incubation with the MTT-containing medium for 4 h. After lysis with dimethyl sulfoxide, the absorbance of lysates was determined at 570 nm by using a microplate reader.

Six-hundred Huh7 cells and 200 cells of NIH3T3-W4P were seeded onto 6-well plates and incubated for 24 h. The NIH3T3 and Huh7 cells were then treated with different concentrations of ACE for 3 and 8 days, respectively. Colonies were fixed with 100% methanol for 10 min and then stained with 0.1% crystal violet for an hour. After washing with PBS, the number of colonies was counted.

The effect of ACE on the phosphorylation status of STAT3 was analyzed by immunoblotting. HepG2 cells were treated with ACE for 3 h prior to treatment with IL-6 (25 ng/ml). After incubation for 25 min, the cells were lysed and the cell lysates were subjected to immunoblotting using anti-STAT3 (Santa Cruz Biotechnology, sc-482), anti-phosphoSTAT3 (Cell Signaling, #9131), and anti-β-actin antibodies (Santa Cruz Biotechnology, sc-47778). To analyze the effect of ACE on the expression of cyclin D2 and P21, HepG2 cells were treated with ACE for 48 h and subjected to immunoblotting using anti-cyclin D2 (Santa Cruz Biotechnology, sc-593) and anti-P21 (Santa Cruz Biotechnology, I2807) antibodies.

To determine the effect of ACE on the mRNA levels of cyclin D1, which are dependent on STAT3 transcriptional activity, RT-qPCR assays were performed. HepG2 cells and NIH3T3-W4P cells were treated with the increasing concentrations of ACE for 4 and 8 h, respectively. Total RNA was extracted using an RNeasy RNA extraction kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized from RNA using Superscript III reverse transcriptase (Invitrogen) with oligo20(dT) primers and analyzed by real-time PCR.

Effect of ACE on Huh7 cell migration was investigated by performing a scratch wound assay. The confluent monolayer of Huh7 cells in a 6-well plate was wounded with a sterile pipette tip and incubated with increasing concentrations of ACE for 48 h. The cells were observed and images were obtained at 24 and 48 h after the treatment.

Cell migration assay was performed as previously described (25). Briefly, after being treated with ACE (0, 25, 50, 100, and 200 µg/ml) for 24 h, Huh7 cells were harvested and seeded on a Boyden chamber (Neuro Probe, Cabin John, MD, USA) at a density of 10⁵ cells per well in a serum-free medium and then incubated for 6 h at 37°C. The migrated cells were fixed with 100% methanol and stained with 5% Giemsa. Cells were counted under a light microscope.

Statistical analysis was performed by SigmaPlot 10.0. Data are shown as means ± SD, and group differences were analyzed using paired Student's t-test. p<0.05 was considered as statistically significant.

AC has been well known for its therapeutic effect in liver diseases and several previous studies suggested its antitumor activity. Here, we evaluated the anticancer activity of ACE on HCC by examining its effect on HCC cell proliferation by MTT assay. ACE displayed significant anti-proliferative effects against Huh7 and HepG2 human hepatoma cell lines in a dose-dependent manner (Fig. 1A and B). To further confirm the anticancer effect of ACE, its effect on the colony-forming ability of HCC cells was examined. As shown in Fig. 2, the colony formation of Huh7 cells was significantly inhibited in the presence of ACE; 100 µg/ml of ACE treatment resulted in 90% reduction of the colony formation. Compared to its direct cell cytotoxicity and the suppression of cell proliferation, ACE exerted potent inhibitory effect on colony forming.

Inhibition of proliferation and colony formation of HCC cells by ACE indicate its anticancer effect against HCC. Given that IL-6-mediated STAT3 activation is implicated in HCC development and progression, the role of ACE in STAT3 activation was examined. IL-6 treatment induced strong phosphorylation of STAT3, which is a hallmark of STAT3 activation in HepG2 cells. Treatment of IL-6-treated HepG2 cells with ACE resulted in marked decrease of both STAT3 and phosphorylated STAT3, indicating that ACE is capable of suppressing the action of STAT3 (Fig. 3A). Since STAT3 upregulates the transcription of cell cycle related genes including cyclin D1 and p21 and enhances cell cycle progression, the effect of ACE on the amounts of cyclin D1 and p21 was examined. Consistent with the suppression of STAT3 phosphorylation by ACE, synthesis of cyclin D1 mRNA as well as protein levels of cyclin D2 and p21 were also decreased by ACE treatment (Fig. 3B and C).

In our previous study, we showed that male-specific W4P mutant HBV LHB drives HCC tumorigenesis by activating the IL-6/STAT3 signaling pathway (4). Thus, we evaluated whether ACE suppresses the STAT3 activation induced by W4P LHB. NIH3T3 cells stably expressing W4P LHB (NIH3T3-W4P) showed strong STAT3 phosphorylation compared to control NIH3T3 cells and the phosphorylation was decreased by ACE (Fig. 4A). The activity of ACE was dose-dependent and 100 µg/ml of ACE was sufficient to lower the phosphorylation to significant extent (Fig. 4B). In line with the results, cyclin D1 mRNA level of NIH3T3-W4P was also decreased in a dose-dependent manner by ACE treatment, further confirming that ACE suppresses the transcriptional activity of STAT3 (Fig. 4C). It has been shown that W4P LHB confers in vitro colony forming ability and in vivo tumor forming capability in nude mice to NIH3T3 cells by activating the STAT3 signaling pathway (4). Since ACE suppresses STAT3 activation in NIH3T3-W4P cells, we evaluated whether ACE suppresses the colony-forming capability of NIH3T3-W4P cells. As shown in Fig. 4D, ACE treatment decreased the number of colonies in a dose-dependent manner, and 250 µg/ml of ACE almost completely abolished the colony-forming ability of NIH3T3-W4P cells. These results suggest that ACE is capable of suppressing tumorigenesis induced by HBV mutations and subsequent inflammatory responses including STAT3 activation in addition to HCC cell proliferation.

STAT3 has been implicated in cancer cell migration and invasion through the upregulation of pro-migratory genes including MMP-2 (26). In the absence of ACE, Huh7 cells migrated along the edge and repaired approximately 30% of the wound after 48 h. Significant suppression of cell migration and wound recovery were observed in the presence of ACE and the inhibitory activity was concentration-dependent (Fig. 5A and B). Cell migration assay using Boyden chamber showed similar result. Treatment with 200 µg/ml of ACE suppressed over 95% of cell migration further confirming the suppressive activity of ACE on tumor cell migration (Fig. 6).