Cell lines and experimental animals
Raji and Molt4 cell lines were cultured at 37°C in 5% CO2 in RPMI 1640 with 10% heat-inactivated FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. The BALB/C nude mice (4–5 weeks of age, non-fertile, female and 18–20 g each) were purchased from the Experimental Center of Southern Medical University. All animal study procedures were approved by the Southern Medical University Animal Care and Use Committee.
Establishment of non-Hodgkin lymphoma animal model and treatment
Mice were injected subcutaneously on the back with 1×107 Raji cells suspended in 0.2 mL sterile PBS. Nude mice were randomly divided into 3 groups, and with 6 mice in each group. The day mice received cell injection was counted as day 0. Mice in the control group were administrated with normal saline by oral gavage once per day for 10 days from +8 day to +12 day and +15 day to +19 day while those in DS and DS/Cu groups were given DS (2.88 mg/20 g per morning) and DS/Cu (DS: 2.88 mg/20 g per morning, Cu: 0.012 mg/20 g per afternoon) respectively. The dosage used here was determined by our preliminary experiment. Length and width of the tumor were measured with a vernier caliper from the beginning of treatment every other day with the volume being calculated using the formula V = a × b2/2 and growth curve being documented. The weight of each mouse was also recorded every other day. Mice were sacrificed on day 20, and the tumors were removed for western blotting analysis.
MTT cytotoxicity assay
Drug cytotoxicity was determined using the colorimetric MTT assay. Briefly, cells (1 × 105 cells/well) were plated into 96-well plates containing 100 μl of the growth medium in the absence or presence of increasing concentrations of drugs at 37°C in 5% CO2 for 24 h and 48 h, MTT (50 μl/well, 5 mg/ml in PBS) was then added and incubated for 4 h at 37°C. The cells were further treated with 100 μl DMSO to dissolve the dark blue crystals of formazan and the absorbance was measured at 570 nm in a microplate reader (ELX800, BioTEK, USA). All experiments were repeated at least three times with triplicate in each experiment. The cytotoxicity of DS or DS/Cu was analyzed and concentration-effect curves were generated as a plot of the fraction of affected cells versus drug concentration. Growth inhibition was expressed as a percentage of the untreated controls that were processed simultaneously. The IC50 was defined as the concentration that inhibited cell growth by 50% (50% reduction of absorbance) compared with untreated controls.
Flow cytometric analysis of apoptotic cells
Cells (3 × 105) cultured in 25 cm2 flasks and exposed to different treatments for 6 h, 12 h, or 24 h were harvested respectively, washed twice with ice-cold PBS and then re-suspended in 500 μl binding buffer. The cells were further incubated with Annexin V-FITC and Propidium Iodide for 15 min at room temperature in the dark according to the manufacturer’s instructions. The stained cells were analyzed by flow cytometry using FACS Calibur (BD Biosciences, Oxford, UK) and Cell Quest (BD Biosciences) software.
Determination of ROS production
ROS production in cells was determined utilizing 2’,7’- dichlorodi hydro fluorescein diacetate (DCFDA). Briefly, 4 × 105/ml cells were taken in a culture dish and treated with DS or DS/Cu for 6 h, 12 h or 24 h. After the treatment, cells were collected and DCFDA (Sigma-Aldrich, Dorset, UK) was added to the cell suspension at a final concentration of 10 μM. After 30 minutes of incubation in the dark at 37°C, cells were centrifuged and the pellet was washed twice with ice-cold PBS. The pellet was then resuspended in FACS buffer and the fluorescence was analyzed with FACS Calibur (BD Biosciences, Oxford, UK) and CellQuest (BD Biosciences) software. DCFDA fluorescence intensity was measured in FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The percentage of ROS producing cells was calculated by counting only those cells, which produced high levels of ROS.
Western blot analysis
Whole protein (50 μg/lane) from each sample was resolved in 10% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a PVDF membrane (Millipore, UK) and blotted with various antibodies. Non-specific binding was avoided by blocking the nitrocellulose membrane with 5% skimmed milk in TBS-T for 1 h. The 5% skimmed milk in TBS-T was also used to dilute primary (SAPK/JNK, rabbit polyclonal, 1:1000, CST; Phospho-SAPK/JNK, rabbit polyclonal 1:1000, Cell Signaling techonology; Phospho-c-jun, rabbit polyclonal, 1:500, Bioworld Technology Co., Ltd., c-jun, rabbit polyclonal, 1:1000, Santa cruz; P65, rabbit polyclonal, 1:1000, Santa Cruz; Nrf2, rabbit polyclonal, 1:1000) and HRP-conjugated monoclonal secondary (1:5000; Amersham Pharmacia Biotech, NJ) antibodies. The membranes were incubated with the primary antibodies overnight at 4°C and in the secondary antibody for 1 h at room temperature. The quantity of protein loaded was verified by staining the same membranes with anti-β actin antibody (1:2000, Sigma-Aldrich, Dorset, UK). The signals were detected on X-ray films using an ECL Western blotting detection kit (Amersham, Pharmacia Biotech).
Statistical analysis
All results were analyzed by Student’s t-test and ANOVA using SPSS 13.0. The statistical significance was indicated by a p value < 0.05.