HeLa229 human cervical carcinoma cells were obtained from the China Center for Type Culture Collection (Wuhan, China). Berberine hydrochloride was purchased from Xi'an Guanyu Bio-Tech Co., Ltd. (Xi'an, China). Fetal calf serum (FCS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). Trypsin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Amresco, LLC (Cleveland, OH, USA). Penicillin, streptomycin and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). The Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Detection kit was from BioVision, Inc. (Milpitas, CA, USA). All other chemicals and solvents used were of the highest purity grade available.

Cells were cultured in RPMI-1640 medium, supplemented with 10% heat-inactivated FCS, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂.

Cells in the exponential growth phase were harvested, adjusted to 2×10⁴ cells/ml and seeded in 96-well plates (200 μl/well). Following a 24-h incubation at 37°C, the medium was removed and berberine hydrochloride was added to wells in a final concentration range of 3.362–215.168 μmol/l. The plate was incubated for a further 72 h, following which 20 μl 5 mg/ml MTT reagent was added to wells. Subsequent to a 4-h incubation at 37°C, formazan crystals formed by live cells were dissolved with 150 μl DMSO and absorbance was measured at 490 nm using a microplate reader (DG5033A; Nanjing Huadong Electronics Group Medical Equipment Co., Ltd., Nanjing, China). Viability was determined using the following formula: % of growth = (optical density of treated cells/optical density of untreated cells) × 100. The half maximal inhibitory concentration (IC₅₀) values were calculated as the concentration of drug required to inhibit 50% proliferation compared with untreated cells.

Experiments were conducted as described previously (24,25), using an Annexin V-FITC/PI Apoptosis Detection kit. Cells at a density of 1.5×10⁵ cells/ml were incubated with 26.896 or 107.584 μmol/l berberine hydrochloride at 37°C for 48 h. Adherent and floating cells were harvested, washed twice with PBS and suspended in 500 μl of 1X Binding Buffer. Annexin V-FITC (5 μl) and 10 μl PI were added and cells were vortexed and incubated for 5 min in the dark. Cells were visualized immediately using an inverted fluorescence biological microscope XD-101 (Nanjing Jiangnan Photovoltaic Group Co., Ltd., Nanjing, China).

Cells at a density of 1.5×10⁵ cells/ml were incubated with 42.93 or 107.584 μmol/l berberine hydrochloride at 37°C for 24, 48 and 72 h. Apoptosis was detected using the Annexin V-FITC/PI Apoptosis Detection kit, as aforementioned. Cells were analyzed immediately by flow cytometry using an FC 500 (Beckman Coulter, Inc., Brea, CA, USA).

Cells (1.5×10⁵ cells/ml) were incubated with 21.465, 42.93 or 107.584 μmol/l berberine hydrochloride for 48 h. Total RNA was prepared using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol and was reverse transcribed using RevertAid™ Moloney Murine Leukemia Virus Reverse Transcriptase and oligo (dT) primers (Fermentas; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA). qPCR was performed on the resulting cDNA using an ABI 7900HT Fast Real Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and SYBR^® Green Real Time PCR Master mix (Toyobo Co., Ltd., Osaka, Japan). The reaction mixture volume was 25 μl, including 11.2 μl PCR water, 2.5 μl SYBR^® Green Real Time PCR Master mix, 0.5 μl forward primer (10 μM), 0.5 μl reverse primer (10 μM) and 0.3 μl cDNA. Primers were synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China), and sequences are presented in Table I. The cycling conditions were as follows: An initial denaturation step at 94°C for 7 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 63°C for 30 sec and extension at 72°C for 20 sec. Results were analyzed to determine the PCR cycle number that generated the first fluorescence signal over a threshold [quantification cycle (Cq), 10 standard deviations (SDs) over the mean fluorescence generated during the baseline cycles], following which the ΔΔCq method was used to measure relative gene expression (29). Expression of the analyzed genes were normalized to the endogenous reference gene, β-actin.

All experiments were performed in triplicate. Data are expressed as the mean ± SD. Data were analyzed in SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA), using one-way analysis of variance followed by the least significant difference test to compare treatment and control groups. P<0.05 was considered to indicate a statistically significant difference.

The effects of berberine hydrochloride on the viability of HeLa229 human cervical carcinoma cells were evaluated using an MTT assay (Fig. 1). The IC₅₀ for HeLa229 cells at 72 h was 42.93 μmol/l. Berberine hydrochloride inhibited HeLa229 cells in a dose-dependent manner. Cell viability following treatment with 3.362, 6.724, 53.791 and 215.164 μmol/l berberine hydrochloride treatment was 99.56, 93.61, 42.85 and 3.61%, respectively. The results demonstrated that HeLa229 cell viability was reduced following a 72-h incubation with berberine hydrochloride.

Apoptosis of HeLa229 cells was detected using the Annexin V-FITC/Propidium Iodide Apoptosis Detection kit. As presented in Fig. 2, apoptotic cells could be observed clearly by fluorescence microscopy. The cell membranes of early and late apoptotic cells were FITC-positive (green), whereas late apoptotic cells additionally had PI-positive (red) nuclei accompanied by condensed chromatin and apoptotic bodies. Increased numbers of late apoptotic cells were observed following treatment with 107.584 μmol/l, compared with 26.896 μmol/l berberine hydrochloride.

As presented in Fig. 3, no significant differences were observed in the proportions of early apoptotic cells following treatment with 42.93 or 107.584 μmol/l berberine hydrochloride for 24 or 48 h, compared with untreated control cells; or in the proportions of late apoptotic cells following treatment with 42.93 μmol/l berberine hydrochloride for 24 h, compared with control cells. Significant early apoptosis was observed following treatment with 42.93 or 107.584 μmol/l berberine hydrochloride for 72 h, at 2.37 and 8.37% of cells, respectively (all P<0.001). The percentage of late apoptotic cells treated with 42.93 μmol/l berberine hydrochloride for 48 h was greater than for cells treated with 107.584 μmol/l berberine hydrochloride; however, this was reversed at 72 h, at 16.43 and 37%, respectively. The percentage of total apoptotic cells increased markedly from 7.3% in the 42.93 μmol/l treatment group at 48 h to 45.37% in the 107.584 μmol/l treatment group for 72 h. Significant differences were observed in the proportions of intact cells (non-apoptotic live cells) at 24, 48 and 72 h between the three groups (all P<0.001). In addition, compared with control groups, significant differences were detected in the proportions of early and late apoptotic cells at 72 h [all P<0.001, except early apoptotic cells of the 42.93 μmol/l treatment group (P=0.007)], and in the percentage of total apoptotic cells at 24 (42.93 μmol/l, P=0.001; 107.584 μmol/l, P<0.001), 48 and 72 h (all P<0.001). Compared with the 42.93 μmol/l berberine hydrochloride treatment group, the 107.584 μmol/l berberine hydrochloride treatment group revealed significant differences (P<0.001) in the percentage of intact cells at all time points and early and late apoptotic cells at 72 h, and in the percentage of total apoptotic cells at 24 h (P=0.014) and 72 h (P<0.001). These results suggest that berberine hydrochloride induced apoptosis of HeLa229 cells in a dose- and time-dependent manner.

p53, Bcl-2 and cox-2 mRNA expression levels in HeLa229 cells were assessed by RT-qPCR, following treatment with 21.465, 42.93 or 107.584 μmol/l berberine hydrochloride for 48 h (Fig. 4). Berberine hydrochloride upregulated mRNA expression levels of p53, whereas mRNA expression levels of Bcl-2 and cox-2 were downregulated in a dose-dependent manner. mRNA expression levels of p53 increased from 1.287- to 2.57-fold relative to control, whereas mRNA expression levels of cox-2 decreased from 0.856-to 0.545-fold, and Bcl-2 decreased from 0.962- to 0.775-fold. Significant differences were observed in p53 mRNA expression levels between treated (21.465 μmol/l, P=0.025; 42.93 μmol/l, P<0.001; 107.584 μmol/l, P<0.001) and untreated control cells, and in cox-2 mRNA expression levels between cells treated with 42.93 (P=0.039) or 107.584 (P=0.002) μmol/l berberine hydrochloride and control cells.