Materials & Methods
Cell Culture & Reagents
Human HL60 and K562 leukemia cells were obtained from the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences (Tianjin, China). DOX-resistant human HL60/A cells and IM-resistant human K562/G cells were established from the parental HL60 and K562 cells in our laboratory as described previously.[26,27] These cells were cultured in RPMI 1640 growth medium containing 10% fetal calf serum and 1% penicillin–streptomycin at 37°C in 5% CO2. TPL, obtained from Sigma (MO, USA), was dissolved in dimethyl sulfoxide and diluted to various concentrations with culture medium before being added into culture. Imatinib mesylate (STI571, kindly provided by Novartis Pharmaceuticals, Basel, Switzerland) was dissolved in dimethyl sulfoxide and diluted to various concentrations with culture medium before being added into culture. All of the stock solutions were stored in aliquots at -20°C.
Methyl Thiazole Tetrazolium Bromide Proliferation Cytotoxicity Assay
Drug cytotoxicity was determined using the colorimetric methyl thiazole tetrazolium bromide (MTT) proliferation assay. Briefly, cells (1 × 105 cells/well) were plated into 96-well plates (Costar, MA, USA) containing 100 µl of the completed 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 phosphate buffered saline) was then added and incubated for 4 h at 37°C. The cells were further treated with 0.1 N acidic isopropanol to dissolve the dark blue crystals of formazan and the absorbance was measured at 570 nm in an ELISA reader. All experiments were repeated at least three times. Each experiment was carried out in triplicate. The cytotoxicity of DOX or IM alone and in combination with TPL was analyzed. 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.
Data from MTT assays were analyzed using the method of Chou and Talalay. This method is based on the median-effect equation for dose–effect relationship: fa/fu = (D/Dm)m, where D is the dose, Dm is the dose required for 50% effect (IC50), fa is the fraction affected by dose D (e.g., 0.6 if cell growth is inhibited by 60%), fu = 1 - fa (or unaffected fraction) and m is a coefficient of the sigmoidicity of the dose–effect curve. Dm and m are easily determined by the median-effect plot: x = log(D) versus y = log(fa/fu), which is based on the logarithmic form of the equation fu = 1 - fa. m is the slope and Dm is the antilog of the x-intercept.
Mean values from three independent experiments were used to calculate the combination index (CI). A CI is then determined on the basis of the isobologram analysis for mutually exclusive effects: CI = (D)1 /(Dx)1 + (D)2/(Dx)2 where (Dx)1 and (Dx)2 are the concentrations of drug 1 and 2 alone required to inhibit cell growth by x% and (D)1 and (D)2 are the drug concentrations in combination treatments that also inhibit cell growth by x%. (Dx)1 and (Dx)2 values can be determined by a rearrangement of equation D = Dm (fa/[1 - fa])1/m. Different values of CI may be obtained for solving the equation for different values of fa (e.g., different degrees of inhibition of cell growth). CI values of <1, 1 and >1 indicate synergism, additive effect and antagonism, respectively.
Flow Cytometric Analysis of Apoptotic Cells
Cells (1 × 106) cultured in 25 cm2 flasks and exposed to different treatments for 24 h were harvested, washed twice with ice cold phosphate buffered saline and then resuspended in 500 µl binding buffer. The cells were further incubated with Annexin V-fluorescein isothiocyanate 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.
Western Blot Analysis
The cytoplasmic protein (50 µg/lane) from each sample was resolved in 10% SDS-PAGE, transferred to a polyvinylidene fluoride membrane (Millipore, UK) and blotted with the various antibodies. Nonspecific binding was avoided by blocking the polyvinylidene fluoride membrane with 5% skimmed milk in tris-buffered saline-tween for 1 h. The 5% skimmed milk in tris-buffered saline-tween was also used to dilute primary (HIF-1α, rabbit polyclonal, 1:1000, Millipore; CXCR4, rabbit polyclonal, 1:1000, Millipore; Nrf2, rabbit polyclonal, 1:1000, cell signaling technology) and horseradish peroxidase-conjugated monoclonal secondary (1:5000; Amersham, GE healthcare Life Science, Buckinghamshire, UK) 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 GAPDH antibody (1:2000, Sigma-Aldrich, Dorset, UK). The signals were detected on x-ray films using an enhanced chemiluminescence western blotting detection kit (Amersham, GE healthcare Life Science).
Real-time PCR Analyses
Cells were lyzed with the Trizol reagent and total RNA was isolated with chloroform and isopropyl alcohol. A total of 1 µg RNA was subjected to reverse transcription with the reverse transcription reagent kit (Takara, Dalian, China) according to the manufacturer's instructions. Then the cDNA was amplified by real-time (RT)-PCR with the SYBR® Prime Script RT-PCR kit (Takara) with the following primers (synthesized by Takara): GAPDH: 5'-GAA GGT GAA GGT CGG AGT C-3' (forward), 5'-GAA GAT GGT GAT GGG ATT TC-3' (backward); HIF-1 α: 5'-CTC AAA GTC GGA CAG CCT CA-3' (forward), 5'-CCC TGC AGT AGG TTT CTG CT-3' (backward); CAIX: 5'-CTT GGA AGA AAT CGC TGA GG-3' (forward), 5'-TGG AAG TAG CGG CTG AAG TC-3' (backward); BNIP3: 5'-TGC TGC TCT CTC ATT TGC TG-3' (forward), 5'-GAC TCC AGT TCT TCA TCA AAA GGT-3' (backward); VEGF: 5'-CTA CCT CCA CCA TGC CAA GT-3' (forward), 5'-CCA CTT CGT GAT GAT TCT GC-3'(backward); HO-1: 5'-TTG CCA GTG CCA CCA AGT TC-3' (forward); 5'-TCA GCA GCT CCT GCA ACT CC-3' (backward); GSR: 5'-CCT GAT CGC CAC AGG TGG TA-3' (forward); 5'-CTG CCA TCT CCA CAG CAA TGT AA-3' (backward); NQO1: 5'- GTG GCA GTG GCT CCA TGT ACT C-3' (forward); 5'-GAG TGT GCC CAA TGC TAT ATG TCA G-3' (backward); Nrf2: 5'-TGG GCC CAT TGA TGT TTC TG-3' (forward); 5'-TGC CAC ACT GGG ACT TGT GTT TA-3' (backward). The alteration of mRNA expression in cells was assessed by 2−ΔΔCt method.
All the experiments were repeated at least three times. Reported values are presented as mean ± standard deviation. The significance of differences between experimental conditions was determined by the two-tailed Student's t-test. Mean values of different groups were compared using one-way ANOVA. The level of significance was p < 0.05.
Pharmacogenomics. 2013;14(11):1305-1317. © 2013 Future Medicine Ltd.