Hep3B cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in DMEM containing 10% fetal bovine serum and 1% antibiotics at 37°C in a 5% CO₂ incubator. Curcumin, TPA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI) and BIO were obtained from Sigma-Aldrich (St. Louis, MO, USA). LY294002 was purchased from Tocris Bioscience (Bristol, UK).

Cells seeded in 12-well plates at 1×10⁵ cells/ml were incubated with curcumin for indicated times and concentrations. Respective medium was removed and then incubated with 20 μl MTT solution (5 mg/ml MTT in PBS) for 1 h. Converted purple formazan dye from MTT was solubilized in DMSO and optical densities were measured at 595 nm.

Cells treated with curcumin at various concentrations (10, 20 and 40 μg/ml for 24 h) were harvested, washed with phosphate-buffered saline (PBS) and fixed in 70% cold ethanol. Following washing with PBS, cells were resuspended in PBS and incubated with RNase A and PI. Cells were analyzed with flow cytometry.

Hep3B cells were grown to 90% confluence in a 6-well plate at 37°C in a 5% CO₂ incubator. A wound was created by scratching cells with a sterile 200 μl pipette tip. Cells were washed with PBS to remove floating cells and then added to a medium. The distance between wound edges was measured at a fixed region. Images of the wound were captured under a microscope (magnification, ×100).

Cells were washed with PBS and lysed with RIPA lysis buffer [50 mM Tris-HCl (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl and 1 mM PMSF]. Protein concentrations were determined using the Bradford assay. All samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. The membrane was incubated overnight with primary antibodies. Secondary antibodies, conjugated to horseradish peroxidase, against IgG were used. Proteins were visualized by enhanced chemiluminescence (Intron Biotechnology, Seong nam, Korea) and detected using the LAS4000 chemiluminescence detection system (Fujifilm, Tokyo, Japan).

Hep3B cells (2×10⁶) were inoculated subcutaneously in 4-week nu/nu mice at the left flank. After 1 week, mice were administered curcumin or PBS (control) by injection. Tumor size was measured in two perpendicular diameters by using a caliper every 3 days and tumor volume was calculated using the following formula: Volume = 1/2 × (length × width²).

Tumor specimens from mice were fixed in 10% formaldehyde, embedded in paraffin and sectioned into 5-μm thick slices. Sections were deparaffinized with xylene and dehydrated with 98% ethanol. Serial sections were stained using standard immunoperoxidase techniques with primary antibodies against CD31. For epitope retrieval, specimens were microwave treated for 25 min prior to incubation with primary antibody. Pre-immune serum was used as a negative control for immunostaining and positive staining was visualized with diaminobenzidine, followed by a light counterstaining with hematoxylin. Sections were evaluated by a pathologist, determining stain intensity and percentage of reactive cells. Representative images were captured. All animal experiments were approved by the Ethics Committee for Animal Experimentation of the Hannam University (Daejeon, South Korea).

Cell viability data were statistically analyzed using unpaired t-tests (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To examine the effects of curcumin on Hep3B cell proliferation, cells were seeded in 12-well plates at a density of 1×10⁵ cells/well. The effect of curcumin on cell viability was determined by MTT assay. Fig. 1A demonstrates significant inhibition of cell proliferation in curcumin-treated cells in a dose- and time-dependent manner, compared with control cells. Cell cycle analysis revealed that curcumin induced dose-dependent apoptosis. The percentage of apoptotic cells (sub-G₁ peak) increased from 19.5% (control) to 35.53% (curcumin 20 μM) and 32.15% (curcumin 40 μM) in the Hep3B hepatocarcinoma cells (Fig. 1B). To determine the mechanism of curcumin-induced apoptosis, the expression levels of apoptotic proteins, including procaspase-3, PARP, Bax and Bcl-2, were detected by western blot analysis. Activation of caspase-3 plays a critical role in apoptosis, as it proteolytically cleaves PARP. In Hep3B cells treated with curcumin, the levels of procaspase-3, PARP and Bcl-2 decreased and Bax increased.

The Wnt signaling pathway plays a critical role in tumor cell growth and survival in hepatocarcinoma. Therefore, the inhibitory effect of curcumin on Wnt signaling in Hep3B cells was determined by analyzing the levels of β-catenin and GSK3β, which play key roles in the pathway. Previous studies have demonstrated that GSK3β is inactivated by Akt and regulated by mTOR (25). In addition, Akt and mTOR are associated with cell survival and growth. Therefore, levels of β-catenin, p-GSK3β, p-Akt and p-mTOR were assessed by western blot analysis. As demonstrated in Fig. 2A, curcumin increased phosphorylation of GSK3β, Akt and mTOR and thus, the level of β-catenin, in a dose-dependent manner. To explore the therapeutic effects of curcumin, hepatocarcinoma tumors were established in nude mice and tumors were treated by injecting curcumin. As revealed in Fig. 2B, tumor volume of the curcumin-treated group was significantly reduced compared with that of the control group. No significant differences in the body weight of mice were observed between control and curcumin-treated groups. Furthermore, curcumin was observed to induce antitumor activity by suppression of Wnt signaling molecules. Fig. 2C demonstrates that β-catenin, p-mTOR, p-Akt and p-GSK3β expression was significantly decreased in the curcumin-treated group compared with the control. To further correlate these observations, in vivo tumor therapeutic effects were assessed in vitro. Thus, the expression levels of biomarkers were assessed in tumor tissues by immunohistochemical analysis. The results indicated that CD31-positive microvessels were significantly suppressed following curcumin treatment (Fig. 2D).

To investigate the effect of curcumin on TPA-induced Wnt signaling activation and cell migration, wound healing assays and western blot analysis were performed in Hep3B cells. Western blot analysis demonstrated that TPA increased β-catenin and phosphorylation of mTOR, Akt and GSK3β, and that curcumin inhibited TPA-induced Wnt signaling pathway activation at 6 h in a concentration-dependent manner (Fig. 3A and B). As demonstrated in Fig. 3C, cell migration was increased in cells treated with TPA. However, the number of migrated cells was significantly decreased in the group treated with curcumin alone and the group pre-treated with TPA and then followed with curcumin for 48 h (for 12 and 24 h; data not shown).

Western blot analysis was used to clarify the mechanism of curcumin-mediated suppression of the Wnt signaling pathway in Hep3B cells by the suppression of specific molecules. The results indicate that curcumin significantly suppressed the expression of p-mTOR, p-Akt, β-catenin and p-GSK3β. Pre-treatment using LY294002 and BIO, specific PI3K and GSK3 inhibitors, respectively, followed by curcumin treatment, effectively reduced β-catenin and phosphorylation of mTOR, Akt and GSK3β (Fig. 4).