The human breast cancer cell lines, MCF-7 and MDA-MB-468, were obtained from the American Type Culture Collection (ATCC; Rockville, MA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Rockville, IN, USA) supplemented with 10% fetal bovine serum (FBS; Haoyang Biological Manufacture Co., Ltd., Tianjin, China), 100 U/ml penicillin and 100 μg/ml streptomycin. All cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂. All the cells used were passaged for >4 months. The identities of these cell lines were validated by short tandem repeat (STR) profiling generated by using the Promega PowerPlex^® 1.2 system. The STR profiles for these cell lines matched their known ATCC fingerprints.

Antibodies against Ku70, Ku86 and RAD51 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse monoclonal antibody against β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-γ-H2AX antibody was purchased from Millipore (Billerica, MA, USA). HRP-labeled secondary antibodies and rhodamine-labeled secondary antibody were purchased from KPL (Gaithersburg, MD, USA). Berberine was purchased from Sigma-Aldrich and dissolved in 100% dimethyl sulfoxide (DMSO) to obtain stock concentrations.

Clonogenic assays were used to assess the survival and proliferation potential of the cells treated with berberine and/or ionizing radiation (IR). The cells were treated with the vehicle control (DMSO) or 15 μM berberine for 24 h. The cells were then irradiated using a Faxitron Cabinet X-ray System (Faxitron X-ray Corp., Wheeling, IL, USA) to deliver the indicated doses (0, 1, 2, 3 and 4 Gy) at room temperature. The X-rays were filtered through a 0.5-mm aluminum filter resulting in a dose rate of 0.4 Gy/min. The cells were trypsinized, suspended in complete medium, counted and replated in 60-mm tissue culture dishes. After irradiation, serial dilutions of irradiated cells were plated immediately. After incubation at 37°C in a humidified atmosphere of 5% CO₂ for 14–21 days to allow the formation of macroscopic colonies, the plates were fixed with methanol and stained with Giemsa. Colonies containing at least 50 cells in size were counted. The fraction surviving a given X-ray dose was calculated based on the survival of non-irradiated cells treated with the vehicle or berberine. Survival (S) data after a radiation dose (D) were fit by a weighted, stratified, linear regression according to the linear-quadratic formula S(D) = S(0) exp(-αD - βD²). The α and β terms in this equation and their ratios were used to describe survival curve characteristics and to classify the cellular response to radiation (23).

Cells were harvested with trypsin, washed with phosphate-buffered saline (PBS) and then stained with buffer including 50 μg/ml propidium iodide (Sigma-Aldrich) for 30 min at room temperature. For fluorescence-activated cell sorting (FACS) analysis, data were collected using a FACSCalibur (BD Bioscience, San Jose, CA, USA) flow cytometer and analyzed by ModFit (Verity, Topsham, ME, USA). The cell-cycle distribution was evaluated by counting >20,000 cells for each sample.

Cells were grown on coverslips in 6-well plates and treated with berberine 15 μM and/or X-ray. At specific times, the medium was aspirated, washed with PBS 3 times and the cells were fixed in 4% paraformaldehyde for 15 min at room temperature, followed by treatment with 0.2% Triton X-100 for 5 min. The cells were then washed with PBS twice and then blocked with 10% normal goat serum in PBS for 50 min, following which mouse anti-γ-H2AX antibody (Millipore) was added at a dilution of 1:200 and incubated overnight at 4°C. The cells were then washed 3 times with PBS before being incubated in the dark with a rhodamine-labeled secondary antibody (KPL) at a dilution of 1:100 in 1% goat serum albumin in PBS for 60 min. The secondary antibody solution was then aspirated and the cells were washed 4 times with PBS. The cells were then incubated in the dark with 4′,6-diamidino-2-phenylindole (1 μg/ml) in PBS for 5 min and coverslips were mounted with an antifade solution (Molecular Probes, Eugene, OR, USA). The slides were then examined on a Leica fluorescent microscope. Images were captured by a charge coupled device camera. For each treatment condition, γ-H2AX foci were counted in at least 100 cells from randomly captured images.

The cells were washed twice with cold PBS and lysed on ice in RIPA buffer [1X PBS, 1% NP 40, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 0.5% sodium deoxycholate and 1 mM sodium orthovanadate] with protease inhibitors and quantified by the BCA method (24). Nuclear and cytosolic extracts were prepared with a nuclear/cytosol fractionation system according to the manufacturer’s instructions. Equal amounts of protein (30–50 μg) were separated by SDS polyacrylamide gel electrophoresis, electrotransferred onto polyvinylidene fluoride membranes (Immobilon-P; Millipore) and blocked with 5% non-fat dry milk in Tris-buffered saline, pH 7.5 (100 mM NaCl, 50 mM Tris and 0.1% Tween-20). The membranes were immunoblotted overnight at 4°C with anti-Ku70, anti-Ku86 and anti-RAD51 monoclonal antibodies (1:200; Santa Cruz Biotechnology, Inc.), and anti-β-actin monoclonal antibody (1:5,000; Sigma-Aldrich), followed by their respective horseradish peroxidase-conjugated secondary antibodies. Signals were detected by enhanced chemiluminescence. β-actin was used as the endogenous control.

The data are presented as the means ± SD and analyzed with Microsoft Excel analysis tools and SPSS statistics 17.0 software. All experiments were repeated independently 3 times. The radiation dose survival curves were analyzed by weighted, stratified, linear regression. Differences between individual groups were analyzed by a paired t-test. P-values of <0.05 were considered to indicate statistically significant differences.

In the MCF-7 cell line (Fig. 1), clonogenic assay revealed that berberine pre-treatment (15 μM, 24 h) reduced the surviving fraction at 2 Gy (SF2) of the irradiated cells from 31.2±0.8 to 20.5±3.9% in the cells treated with radiation alone. The combination treatment caused a reduction of approximately 30% in the SF2 (P=0.002). The data were further analyzed according to the linear quadratic model; the α and β components were 0.552±0.050/Gy and −0.002±0.014/Gy² for the cells treated radiation alone, and 0.758±0.104/Gy and −0.007±0.033/Gy² for the cells treated with the combination treatment, respectively, leading to survival curves which were significantly different (P<0.001) as tested with the linear regression analysis. A similar response was observed in the other human breast cancer cell line, MDA-MB-468, with the SF2 being reduced to 20.1±0.6% when the irradiated cells were pre-treated with berberine at 15 μM for 24 h, in comparison with 33.1±3.1% for the cells treated with radiation alone (P<0.01). The α and β components were 0.538±0.058/Gy and −0.009±0.016/Gy² for the radiation group, and 0.902±0.07/Gy and −0.034±0.021/Gy² for the combination treatment group, respectively. According to the linear regression analysis, a statistical difference between 2 groups was obtained (P<0.001).

We performed flow cytometry analysis of the cells treated with 15 μM berberine or DMSO for 24 h by propidium iodide staining to evaluate the effect of berberine treatment on the cell cycle progression of human breast cancer cells. Berberine treatment induced an increase in the proportion of cells in the G1 phase (77.5±2.1 vs. 57.1±0.5%) and a marked decrease in the proportion of cells in the S phase (21.3±1.2 vs. 40.0±1.9%) in comparison with the control MCF-7 cells (Fig. 2). The same results were observed in the MDA-MB-468 cells. The G0/G1 fraction of the cells treated with berberine increased from 55.4±1.5 to 69.5±2.9%, compared to the control cells, and the fraction of the cells treated with berberine in the S phase decreased to 14.4±1.0% in comparison with the control cells (34.1±0.9%). When the MCF-7 cells were exposed to 6 Gy irradiation alone for 8–24 h, a significant cell cycle arrest in the G2/M phase was observed, with a decrease in the percentage of cells in the G0/G1 phase and S phase. Following pre-treatment with berberine, the radiation-induced G2/M phase arrest did not occur within 24 h after irradiation, with an increase in the percentage of cells in the G0/G1 phase compared with irradiation alone.

H2AX, a variant of the core histone H2A family, contains a unique SQ motif within its C-terminal tail that is highly conserved from plants to humans, suggesting a crucial role of this variant throughout evolution. The phosphorylated form of H2AX was termed γ-H2AX, as it was first observed in cells exposed to γ-rays. The formation of γ-H2AX in response to DNA DSBs provides the basis for a sensitive assay for DNA damage. In the MCF-7 cells, we determined the levels of DSBs by immunofluorescence staining of γ-H2AX foci at different time-points (0, 0.5, 4 and 12 h) after exposure to X-rays. In the MCF-7 cells not pre-treated with berberine, the majority of the γ-H2AX foci cleared 4 h following exposure to 1.0 Gy of X-ray radiation (Fig. 3). By contrast, a delayed clearance of the X-ray-induced γ-H2AX foci was observed in the MCF-7 cells pre-treated with berberine (15 μM). These results indicate that berberine pre-treatment radiosensitizes the cancer cells via the impairment of the repair of X-ray-induced DSBs.

DSBs are primarily repaired by 2 pathways. Ku70 and Ku86 are essential for nonhomologous end joining, whereas RAD51 is a central player in homologous recombination. We examined the expression of Ku70, Ku86 and RAD51 by western blot analysis to assess whether the levels of these 3 proteins were altered in human breast cancer cells pre-treated with berberine. As shown in Fig. 4, there were no obvious changes in the levels of Ku70 and Ku86, but the levels of RAD51 were significantly decreased in the MCF-7 and MDA-MB-468 cells treated with 15 μM berberine for 24–48 h. In the MCF-7 cells treated with 15 μM berberine for 24 h prior to 6 Gy irradiation at the indicated time-points (0, 2, 6 and 24 h), the levels of Ku70 and Ku86 protein had no obvious change, but the level of RAD51 protein had decreased significantly. However, these changes were not observed in the cells treated with irradiation alone. In the MDA-MB-468 cells the same results were observed.