Materials
TMRM Reagent, CellROX™ Green Reagent were purchased from Invitrogen (Life Technologies, CA, USA). Bovine serum albumin (BSA), RNase A, propidium iodide (PI), carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD-FMK), Dihydroethidium (DHE), ATP Assay Kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). MEK Inhibitor U0126 was purchased from Promega (Madison, WI, USA). Cell counting Kit-8 (CCK-8) was purchased from Targetmol (Shanghai, China). Annexin V-FITC/PI apoptosis detection kit was purchased from Fremont (CA, USA). The antibody specific for GAPDH was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies specific for phospho-ERK1/2 (T202/Y204), ERK1/2, puma, Bax, Bcl-xL, Caspase-3, Caspase-9 and PARP were purchased from were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibodies specific for total OXPHOS rodent WB antibody cocktail (Mouse) was purchased from Abcam (Cambridge, MA, USA). Secondary horseradish peroxidase (HRP) conjugated donkey anti-rabbit and donkey anti-mouse were obtained from Invitrogen (Life Technologies, CA, USA).
Preparation of Aqueous Extracts of Toona Sinensis Leaf (TSL)
The TS leaves used in this preparation were obtained from TS grown in Tuku (Yunlin County, Taiwan) and were picked and washed thoroughly with water. A voucher specimen (FY-001) was characterized by Dr. Horng-Liang Lay, Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung County, Taiwan, and deposited at Fooyin University, Kaohsiung. Permissions were also obtained by Dr. Horng-Liang Lay. In brief, the liquid of TSL was concentrated in a vacuum and freeze-dried to form a powder. The TSL aqueous extracts used in experiments were dissolved in sterile phosphate-buffered saline (PBS; pH 7.4) and filtered using a 0.45-mm syringe filter (Satorius Stedim Biotech Inc., Goettingen, Germany) [29].
Cell culture
Human GBM cell lines (A172 [ATCC CRL-1620; ATCC] and U251 [ormerly known as U-373 MG; ECACC 09063001]) were obtained from American Type Culture Collection, (ATCC, Manassas, VA, USA). The culture of A172 and U251 cells was performed in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (100 units/ml of penicillin and 10 mg/ml of streptomycin; all from Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and incubated in a 5% CO2 incubator at 37 °C with fully humidified conditions. All experiments were performed when cells were in the logarithmic phase of growth.
Cell viability and colony formation assay
An CCK-8 assay was used to measure cell viability. Briefly, GBM cells, A172 (4×103 cells/well) and U251 (4×103 cells/well), were seeded in 96-well plates overnight, and were then incubated with various concentrations of TSL at 37°C for 24, 48 and 72 h. After that, add 1/10 volume of Cell Counting Kit-8 (CCK-8) directly to cells in culture medium. Incubate in a cell culture incubator for 1 to 4 hours at 37°C until the color turns orange. Using a Synergy™ HT Multi-Detection Reader (Bio-Tek Instruments, Winooski, VT) absorbance was measured at 450 nm. In addition, colony formation assays were conducted as described previously [30]. Cells were then rinsed with distilled water, air-dried, and colonies were counted and analyzed using ImageJ software. Then, DMSO was added to dissolve this crystal. The results are expressed as the average colony count ± SE from three independent experiments.
Cell cycle analysis
For cell cycle analysis, cultured A172 and U251 cells, following treatment with increasing concentrations of TSL for 48 h, were collected and washed with PBS (pH 7.2). The cells were resuspended in 85% methanol for overnight at 4 °C and then centrifuged at 1500rpm for 10 min. The resultant pellet was washed twice with PBS, suspended in PBS and incubated with stained with 100 μg/mL propidium iodide (PI, Sigma-Aldrich Co) and RNase (20 Units/mL, final concentration) for 30 min. At least 10,000 cells were counted by a flow cytometer (Attune NxT flow cytometer, Thermo Fisher Scientific), and the data obtained were analyzed using and the data obtained were analyzed using Attune NxT Flow Cytometer Software (Thermo Fisher Scientific).
Apoptosis analysis
Apoptosis of A172 and U251 cells treated with various concentrations of TSL in the presence or absence of z-VAD (10 μM) or U0126 (10 μM) for 48 h was determined by the CF®488A Annexin V and PI Apoptosis kit. Phosphatidylserines exposed on the membrane surface of apoptotic cells were stained with Annexin V-FITC according to the manufacturer's instructions. The late apoptotic (or necrotic) cells were stained with PI. Parallel sets of wells containing non-treated A172 or U251 were regarded as the (+) controls. The results were acquired and analyzed with Attune NxT Flow Cytometer Software (Thermo Fisher Scientific). The population of apoptotic cells was characterized by its high mean fluorescence of Annexin V-FITC in a flow cytometric histogram.
Determination of the intracellular ROS, mitochondrial ROS and Mitochondrial Membrane Potential (ΔΨm)
Intracellular ROS production was detected using CellROX, mitochondrial ROS production was detected using Dihydroethidium (DHE) and ΔΨm production was detected using Tetramethylrhodamine methyl ester (TMRM). Following incubation A172 and U251 cells with the different doses of TSL for 48 h, 5 µM CellROX® green reagent, 250 nM DHE red reagent and 25 nM TMRM at 37 °C for 30 min. Subsequently, the cells were washed in PBS, trypsinized and measured fluorescence intensity by flow cytometry at excitation/emission wavelengths of 485/530 nm, 510/580 nm and 488/570 nm for CellROX, DHE and TMRM, respectively, and the results were analyzed using Attune NxT Flow Cytometer Software.
Cell ATP levels
Cultured A172 and U251 cells, following treatment with increasing concentrations of TSL for 48 h, were collected and washed with PBS (pH 7.2). Cell ATP measurement were conducted as described previously [31]. Then sample was measured with absorbance at 570 nm (A570) or the fluorescence (FLU, lex = 535/ lem = 587 nm) in a microplate reader. For each measurement, a standard curve will be constructed using serial dilutions of ATP stock solution. The sample was converted to nanomolar amounts of ATP accordingly. The results are expressed as the average colony count ± SE from three independent experiments.
Western blot analysis
Cells were seeded into 10 cm dish plates, treated different concentrations of TSL in the presence or absence of z-VAD (10 μM) or U0126 (10 μM) for 48 h. Cell lysis, protein concentration, SDS-PAGE gel and western blotting were performed as described previously [32]. The following antibodies dilutions were used for immunodetection: PARP (1:1,000, Cell signaling, Beverly, MA, USA); caspase-3 (1:1,000, Cell signaling, Beverly, MA, USA); caspase-9 (1:1,000, Cell signaling, Beverly, MA, USA); Bcl-2 (1:1,000, Cell signaling, Beverly, MA, USA); Bax (1:1,000, Cell signaling, Beverly, MA, USA); puma (1:1,000, Cell signaling, Beverly, MA, USA); ERK1/2 (1:1,000, Cell signaling, Beverly, MA, USA); phospho-ERK1/2 (T202/Y204,1:1,000, Cell signaling, Beverly, MA, USA); GAPDH (1:5,000, Santa Cruz biotechnology, Heidelberg, Germany).
Statistical Analysis
Data are presented as mean ± standard deviation. Statistical analyses were performed using one-way analysis of variance. Data were compared using Student’s t-test. The level of statistical significance was set at * p < 0.05, ** p < 0.01, *** p < 0.001.
Statement
All methods were carried out in accordance with relevant guidelines and regulations.