Cell culture and treatment
Human prostate cancer cell lines LNCaP and DU-145 were purchased from Shanghai Cell Bank, and cultured in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in 5% CO2 and 21% O2 or 0.8% O2 in a humidified atmosphere. Exponentially growing cells were detached using 0.05% trypsin-EDTA every 2–3 days. The cells were irradiated in ambient air with 6 MV X-rays at a dose rate of 5.66 Gy/min at room temperature. Berberine hydrochloride (>95%) was purchased from Sigma Aldrich.
Cells in early log phase were trypsinized and plated in 96-well plates at a density of 4,000 cells/well. After 24 h, the medium was removed and replaced with fresh medium supplemented with different concentrations of berberine (20, 50, 100, 150, 200, 250, 300, 400 μM). Cell viability was detected on day 1, 2 and 3 by MTT assay following the manufacturer’s instructions. Briefly, 10 μl of MTT was added into each well to a final concentration of 0.5 mg/ml. After incubation for 4 h, the cell supernatants were removed and DMSO (150 μl) was added to dissolve MTT crystals (formazan). The absorbance of the samples at 490 nm was read using a Bio-Rad microplate reader (model 630; Hercules, CA, USA).
Cells in early log phase were trypsinized and plated in 6-well plates. Then the cells were treated with or without bererine for the indicated time and then subjected to X-rays of 2, 4, 6, 8 Gy at room temperature. The cells were incubated at 37°C for 12 days for LNCaP cells and 14 days for DU-145 cells, fixed with methanol and stained with Giemsa. Finally, the plates were inspected by microscopy and the number of the colonies with at least 50 cells was counted.
Flow cytometry analysis of apoptosis
Cells in early log phase were trypsinized and plated in 6-well plates at a specific density. The cells were treated with or without bererine in normxia or hypoxia for 24 h, then exposed to X-rays (6 Gy). After 3 h, the cells were collected and incubated with Annexin V and propidium iodide for 15 min. The apoptotic cells were detected by flow cytometry using light scatter characteristics (BD Bioscience, Oxford, UK).
Western blot analysis
Total cell lysates were prepared by harvesting cells in protein extraction buffer, and protein concentration was analyzed using the BCA protein assay kit. Equal amounts of proteins were separated on 6% or 10% SDS-polyacrylamide gel, transferred onto nitrocellulose membranes (Schleicher and Schuell Bio-Science). The membranes were blocked with 5% nonfat milk in TBST (Tris-buffered saline, pH 7.4 and 0.05% Tween 20) and incubated with HIF-1α antibody (1:200), VEGF antibody (1:250) and β-actin antibody (1:250) (all from Santa Cruz Biotechnology Inc, CA, USA) overnight at 4°C. The membranes were then incubated with alkaline phosphatase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:2000) for 1 h at room temperature, and the signals were detected by using enhanced chemiluminescence detection kit.
Cells in early log phase were trypsinized and plated in 24-well plates at a density of 4,000 cells/well. The cells were exposed to bererine and/or hypoxia for 24 h. Cells were fixed with methanol at −20°C for 20 min, and washed 3 times with PBS. Next the cells were incubated with goat anti-mouse HIF-1α antibody (1:250) or goat anti-rabbit VEGF antibody (1:250) for 16 h at 4°C, then incubated with anti-mouse-FITC or anti-rabbit-FITC (Jackson, USA) at a dilution of 1:150 for 1.5 h in the dark at room temperature. Finally, cells were mounted onto glass slides and observed under Confocal Laser Scanning Microscope (Zeiss LSM510).
Tumor xenograft mouse models
Animal experiments were approved by Ethics Committee of Nanjing Medical University. Male BALB/C nude mice (4–5 weeks old) were provided by Nanjing Medical University Animal Center. Mice were then subcutaneously inoculated with LNCaP cells (5 × 106 cells in 0.1 ml of PBS) at one site of the right armpit. When the average volume of tumour (visualized as small nodules at the sites of injection) increased to 150 mm3, the animals were randomly grouped into 6 different groups (n = 6): (1) vehicle (PBS), (2) 5 mg/kg berberine, (3) 10 mg/kg berberine, (4) 8 Gy IR, (5) 5 mg/kg berberine plus 8 Gy IR, (6) 10 mg/kg berberine plus 8 Gy IR. The mice in control group were treated with vehicle control, whereas the mice in 2, 3, 5 and 6 groups were given daily intraperitoneal injection of 5 or 10 mg/kg berberine every two days for 6 times. Tumors were irradiated by RS-2000 biological irradiator at a dose of 8 Gy with X-rays (2 Gy/min) delivered 2 h after injection on day 12. Tumor growth was measured every two days, the tumor volume was calculated according to the formula: Tumor volume = [length (L) × width (W)]2/2. The percentage of tumor growth inhibition (TGI) was calculated as follows: TGI (%) = [1-(mean change in the tumor volume in each group/mean change in the tumor volume in the control group)] × 100. The tumor doubling time (DT) was calculated as follows: DT = d × lg2/lg(Vd/V0), where d was the length of time between two measurements, Vd was the volume of the tumor treated with X-ray and V0 was the volume of the tumor before the X-ray.
Data were expressed as mean ± Standard Deviation (SD) of at least three independent experiments, and analyzed by SPSS statistical software system for Windows version 16.0 (SPSS Inc. Chicago, USA). Statistical significance was determined by using Student’s t-test and p < 0.05 was considered significant.