Chemicals and antibodies
AITC was purchased from Sigma (St Louis, MO). Y-27632 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LY294002 and Z-VAD-FMK purchased from EMD Biosciences (La Jolla, CA). Antibodies against Akt, cytochrome c, cofilin, actin, PP1, PTEN and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA); cleaved caspase-3, cleaved caspase-9, phospho-Akt (Ser473), phosphor-Cofilin (Ser3), PI3K, phospho-PI3K and Cox IV were from Cell Signaling Technology (Beverly, MA); ROCK1 and SSH were from Abcam (Burlingame, CA); PP2A was from BD Bioscience (San Jose, CA); PARP was from Biomol (Plymouth Meeting, PA).
U937, HL-60, and Jurkat cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells were cultured at 37°C in a humidified atmosphere and 5% CO2 in air.
After approval by the Southwest Hospital Institutional Review Board (Chongqing, China), peripheral-blood samples were obtained from 17 patients with newly diagnosed or recurrent acute myeloid leukemia (AML) after acquiring informed consent. AML blasts were isolated by density gradient centrifugation over Histopaque-1077 (Sigma-Aldrich Co., St Louis, MO) at 600 g for 15 minutes. Isolated mononuclear cells were counted, re-suspended in RPMI 1640 medium at 8×105/mL for treatment. CD34+ cells from bone marrow mononuclear cells of healthy donors were isolated using the MACS cell isolation kit (Miltenyi Biotec, BG, German) according to the manufacturer’s instructions. After washing and enumerating as described for mononuclear cells, cells were suspended at 8×105/mL prior to treatment.
Apoptosis and mitochondrial transmembrane potential assay
Cells were harvested and apoptosis was analyzed by flow cytometry using the Annexin V/PI staining kit (PharMingen, San Diego, CA) according to the manufacturer’s instructions. Briefly, 1×106 cells were washed twice with phosphate-buffered saline (PBS), and stained with 5 μl of Annexin V-FITC and 10 μl of PI (5 μg/mL) in 1× binding buffer (10 mM HEPES, pH 7.4, 140 mM NaOH, 2.5 mM CaCl2) for 15 min at room temperature in the dark. The apoptotic cells were determined using a Becton-Dickinson FACScan cytoflurometer (Mansfield, MA, USA). Both early apoptotic (Annexin V-positive, PI-negative) and late (Annexin V-positive and PI-positive) apoptotic cells were included in cell death determinations.
A diminished mitochondrial membrane potential (△ψm) was monitored using DiOC6. For each condition, 4 × 105 cells were incubated in 1 mL 40 nM DiOC6 at 37°C in for 15 minutes and subsequently analyzed using a Becton Dickinson FACScan cytofluorometer with excitation and emission settings of 488 and 525 nm, respectively.
Preparation of mitochondrial and cytosolic fractions
Mitochondrial and cytosolic fractions were obtained as previously described . Briefly, cell pellets were washed twice with PBS and resuspended in 5 × buffer A (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 2 mM leupeptin, 1 mM PMSF, 1mM DTT, 2 mM pepstatin, and 250 mM sucrose). Cells were homogenized by passing them through a 22-gauge needle 25 times. The homogenate was centrifuged in three sequential steps: 1000 g, 10,000 g, and 100,000 g. The 10,000 g pellet was considered the “mitochondrial” fraction, and the 100,000 g supernatant the “cytosolic” fraction. These fractions were subjected to Western blot and immunoprecipitation analyses.
G-actin/F-actin assay were performed by using G-actin / F-actin In Vivo Assay Kit (Cytoskeleton, Denver, CO) according to the manufacturer’s instructions. Briefly, cells were lysed with LAS2 buffer (containing lysis, F-actin stabilization buffer, ATP stock solution and protease inhibitor cocktail stock solution) at 37°C for 1 h. Unbroken cells were removed by centrifugation at 2,000 rpm for 5 min. Cell lysates were then centrifuged at 100,000 g for 1 h, finally F-actin in the pellet and G-actin in the supernatant. Samples were mix with 5×SDS sample buffer and then analyzed by western blot with antibody against actin.
Western blot and immunoprecipitation analysis
Cells were lysed in 1× NuPAGE LDS sample buffer supplemented with 50 mM dithiothreitol. The proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and processed for immunoblotting as previously described . For immunoprecipitation analysis, Cells were lysed in 1% NP-40 buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM Na3VO4). Equal quantities of proteins were incubated with primary antibodies at 4°C on a rocking platform. Immune complexes were collected with protein G agarose beads (Pierce Biotechnology, Rockford, IL) followed by several washes in lysis buffer, samples were boiled and then subjected to SDS-PAGE/Western blot.
Cells were collected by centrifugation, resuspend gently in pre-warmed (37°C) staining solution containing 200 nM MitoTracker Red CMXRos (Molecular Probes, Eugene, OR) for 1 h at 37°C, and washed twice with RPMI 1640 medium. After fixed with 3.7% of methanol-free formaldehyde for 15 min, and permeabilized with 0.1% Triton X-100 for 10 min. Slides were blocked with 1% BSA in PBS for 30 min, then incubated with anti-cofilin primary antibody at 4°C overnight, followed by the secondary Alexa 488-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR) for 1 h at room temperature. Cells were incubated with 50 nM MitoTracker Green FM (Molecular Probes, Eugene, OR) after fixation because it can not retained well after fixation. Fluorescent staining of globular and filamentous actin was performed using Fluorescent Deoxyribonuclease I Conjugates and Fluorescent phallotoxins (Molecular Probes, Eugene, OR), respectively, according to the manufacturer's instructions. Images were collected and analysed using Leica scanning confocal microscope (TCS SP2 AOB; Wetzlar, Germany).
Animal studies were conducted according to protocols approved by Third Military Medical University Institutional Animal Care and Use Committee. Nude mice (5 weeks old) were purchased from Vital River Laboratories (VRL, Beijing, China), and inoculated subcutaneously with 2×106 U937 cells into the lower back of each mouse. Mice were randomized into two groups (n=20). Five days after tumor inoculation, Mice were received AITC (50 mg/kg, i.p., five times per week) or an equal volume of vehicle. Tumor size and body weight were monitored per week after treatment, and the survival time of mice was recorded. Tumor tissues from representative mice were fixed in paraformaldehyde, embedded in paraffin, sectioned and processed for hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL) and immunohistochemical analysis.
TUNEL, histological and immunohistochemical assay
TUNEL assay were performed by using an In Situ Cell Death Detection kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Histological and immunohistochemical assay were performed as previously described .
All of the data are expressed as mean ± SD of three individual experiments. Group measurements were compared using a Student’s t-test or analysis of variance (ANOVA). Survival analysis was performed with the Kaplan–Meier method, and significance was calculated using the log-rank test. *P < 0.05 or **P < 0.01 were considered significant.