Berberine (purity, >99%), sulforhodamine B (SRB)and protease inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA). The high-glucose medium, Dulbecco’s Modified Eagle’s Medium (DMEM), was obtained from Gibco-BRL (Carlsbad, CA, USA). The Bradford Protein Assay kit, RIPA lysis buffer and the Annexin V-FITC Apoptosis Detection kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Activated signaling pathways were identified using primary antibodies, including anti-AMPK, anti-phosphospecific AMPK, anti-Akt, anti-phosphospecific Akt, anti-Bax, anti-Bcl-2, anti-cytochrome c, anti-caspase-3 and anti-caspase-9, purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The internal control antibody (anti-β-actin) was also obtained from Santa Cruz Biotechnology, Inc.

The human hepatocelluar carcinoma cell lines (HepG2, SMMC-7721 and Bel-7402) and the normal liver cell line (HL-7702) were purchased from Hsiang-Ya Medical College (Hunan, China). Cells were cultured at 37°C and 5% CO₂ in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 mg/ml). The study was approved by the ethics committee of Sichuan University.

The in vitro cytotoxicity of berberine against HepG2, SMMC-7721, Bel-7402 and HL-7702 cells was assessed using the traditional SRB assay. The SRB assay is routinely used for cytotoxicity determination, based on the measurement of live cell protein content (19). In brief, cells were seeded into 96-well plates at a density of 7,000 cells/well. Following 24 h of culture in DMEM supplemented with 10% FBS, the medium was replaced with DMEM without FBS for 24 h. Subsequently, the cells were treated with berberine at final concentrations of 3.125, 6.25, 12.5, 25, 50 and 100 μM for 24 or 48 h prior to the SRB assay. All experiments were conducted in parallel with controls; at 24 or 48 h, 100 μl of 10% trichloroacetic acid was also added to each well when staining for 30 min, and then the excess dye was removed by washing repeatedly with 1% acetic acid. The protein-bound dye was dissolved in 10 mM Tris Base solution (Santa Cruz Biotechnology, Inc.) for optical density (OD) determination at 570 nm using a microculture reader (Bio-Rad, Richmond, CA, USA). The percentage of viable cells was calculated as follows: (A of experimental group/A of control group) ×100, where A represents the absorbance.

Apoptosis was analyzed by FCM using the Annexin V-FITC Apoptosis Detection kit (Beyotime Institute of Biotechnology), according to the manufacturer’s instructions. Briefly, following treatment with 0, 12.5, 50 and 100 μM berberine for 24 h, HepG2 cells were detached and resuspended in 100 μl binding buffer containing fluorescein isothiocyanate-conjugated annexin V and propidium iodide (PI). Following incubation for 15 min at room temperature in the dark, the cells were analyzed by FCM (BD Biosciences, Franklin Lakes, NJ, USA).

The cells were washed twice with cold phosphate-buffered saline (PBS) and then suspended in RIPA buffer for 30 min on ice. The supernatant was collected as the total protein extract. The protein concentration was estimated by the Bradford Protein Assay kit (Beyotime Institute of Biotechnology), according to the manufacturer’s instructions. Equal amounts of protein (40 μg) were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 5 or 12% separation gel, and molecular weight markers were run simultaneously. The proteins were transferred to nitrocellulose membranes and then blocked for 2 h at room temperature in a solution of 5% milk or bovine serum albumin (BSA). Incubation with the appropriate primary antibodies was performed overnight at 4°C. Membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:2,000 dilution) for 2 h at room temperature with gentle agitation. After washing, bands were visualized by an Enhanced Chemiluminescence (ECL) Western Blotting Detection system (Amersham Pharmacia Biotech, NJ, USA).

All data were expressed as the mean ± SE. The statistical significance of differences in the data was determined by Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the antitumor characteristics of berberine, cell viability was measured by a standard SRB assay following brief exposure to berberine (24 or 48 h). As demonstrated in Fig. 2, berberine significantly reduced cell viability in human hepatoma cell lines in a time- and dose-dependent manner. Subsequently, we predicted the side effects of berberine by exposing the normal hepatic cell line, HL-7702, to the agent. The IC₅₀ values in the HL-7702 cell line (838.4 μM) at 48 h were markedly higher than those of the HCC cell lines (34.5 μM, 25.2 μM and 53.6 μM in the HepG2, SMMC-7721 and Bel-7402 cell lines, respectively). The differences in the IC₅₀ values indicated that berberine was able to selectively decrease the cell viability of HCC cells compared with normal hepatocytes.

To further investigate the inhibition mechanism of berberine in HCC, we selected HepG2 cells for further study. HepG2 cells were treated with 12.5, 25 and 50 μM berberine for 24 h, and apoptosis was detected by flow cytometric analysis with the Annexin V-FITC Apoptotic Detection kit (Beyotime Institute of Biotechnology). Data revealed that the pretreatment of HepG2 cells with berberine significantly increased the number of early apoptotic cells (annexin V⁺ and PI⁻) and late apoptotic cells (annexin V⁺ and PI⁺) compared with the control group, in a dose-dependent manner (Fig. 3A). At a concentration of 50 μM, berberine led to ≤40% apoptosis in HepG2 cells (Fig. 3B). These results suggested that berberine-induced apoptosis in the liver cancer cells contributed to its antiproliferative and cytotoxic effects.

In chemically induced apoptosis, mitochondria play a central role in the commitment of cells to apoptosis through cytochrome c-dependent pathways. To elucidate the molecular mechanism of berberine-induced apoptosis in HCC cells, we examined the expression of proteins associated with apoptosis. HepG2 cells were treated with 12.5 and 50 μM berberine for 24 h. The results demonstrated that creatine kinase induces the release of cytochrome c from mitochondria to the cytosol in dose-dependent manner (data not shown). Caspase-9 and -3, well-known downstream molecules of cytochrome c, were also cleaved in a concentration-dependent manner (Fig. 4A). We further evaluated the effect of berberine on the mitochondrial apoptosis signaling pathway. We found that the expression of Bcl-2 protein, an anti-apoptotic molecule, decreased in a dose-dependent manner, whereas the expression of the pro-apoptotic protein, Bax, was significantly increased (Fig. 4B). Furthermore, the ratio of Bax/Bcl-2 was increased in a dose-dependent manner (Fig. 4C). These results indicated that treatment with berberine leads to a shift from anti-apoptosis to pro-apoptosis by altering the function of the proteins in the Bcl-2 family, which results in the release of cytochrome c from mitochondria, thus inducing caspase-dependent mitochondrial pathway cell apoptosis.

Several reports have demonstrated that activation of AMPK leads to the induction of apoptosis in numerous human cancer cell types (20,21). Therefore, we investigated whether the phosphorylation of AMPK is induced by berberine. HepG2 cells were treated with 12.5 and 50 μM berberine for 24 h. As demonstrated in Fig. 5A, compared with the basal level, berberine significantly stimulated the phosphorylation of AMPK (Thr-172), which increased the ratio of p-AMPK/total-AMPK in dose-dependent manner. The measurements of the phosphorylation pattern of Akt revealed that the pretreatment of HepG2 cells with berberine resulted in a marked elevation in the levels of phosphorylated Akt (Ser-473) relative to the control cells, without significantly altering the total protein levels of Akt (Fig. 5B). These results indicated that AMPK activation is correlated with the induction of apoptosis.