2.1. Chemicals and reagents
ACP-93 was synthesized and kindly provided by our collaborator Dr. Chinpiao Chen (National Dong Hwa University, Hualien, Taiwan). Wortmannin, propidium iodide and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical (St. Louis, MO). 3,3‟-dihexyiloxadicarbocyanine iodide (DiOC6) and 2‟,7‟-dichlorodihydrofluorescein diacetate (H2DCF-DA) were purchased from Calbiochem (San Diego, CA). Annexin V-FITC/PI kit was purchased from BioVision, Inc. (San Francisco, CA).
2.2. Antibodies
Anti-AKT and anti-phospho-AKT (ser473) were purchased from Cell Signaling Technology, Inc. (Boston, MA, USA). Anti-ATF-3 (C-19), anti-bax (N-20), anti-bcl-2 (100), anti-actin (I-19) and anti-survivin (D-8) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-caspase 3 was purchased from BioVision, Inc. (San Francisco, CA). Anti-poly(ADP-ribose) polymerase (PARP) was purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-γ-H2AX and anti-caspase 8 were purchased from BD Bioscience. (Franklin Lakes, NJ).
Antibodies Molecular
Weight Incorporation Source Antibody
dilution
AKT 60 KDa Cell Signaling Technology, Inc. rabbit 1:1000
phospho-AKT
(ser473) 60 KDa Cell Signaling Technology, Inc. rabbit 1:1000
ATF-3 (C-19) 21 KDa Santa Cruz Biotechnology, Inc. rabbit 1:500
Actin (I-19) 42 KDa Santa Cruz Biotechnology, Inc. mouse 1:5000
Bax (N-20) 21 KDa Santa Cruz Biotechnology, Inc. rabbit 1:1000
Bcl-2 (100) 29 KDa Santa Cruz Biotechnology, Inc. mouse 1:1000
Caspase 3 32 KDa BioVision, Inc. mouse 1:1000
Caspase 8 55 KDa BD Bioscience. mouse 1:2000
poly(ADP-ribose)
polymerase (PARP) 116 KDa Cell Signaling Technology, Inc. rabbit 1:1000 phospho-histone
H2AX (ser139) 13 KDa BD Bioscience. mouse 1:1000
Survivin (D-8) 16.5 KDa Santa Cruz Biotechnology, Inc. mouse 1:500
2.3. Cell culture
The securin-wild type and securin-null HCT116 colorectal carcinoma cell lines were kindly provided by Dr. B. Vogelstein of Johns Hopkins University (Baltimore, MD). The HCT116 cells were derived from an adult male colorectal carcinoma. The cells were maintained in McCoy‟s 5A medium. The complete medium was supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 mg/ml streptomycin and sodium bicarbonate. These cells were maintained at 37°C and 5% CO2
in a humidified incubator (310/Thermo, Forma Scientific, Inc., Marietta, OH).
2.4. Cytotoxicity MTT assay
The cells were plated in 96-well plates at a density of 1 × 104 cells/well for 16-20 h. Thereafter the cells were co-treated with or without PI3K/AKT inhibitor (wortmannin) and ACP-93 for 24 h in complete McCoy‟s 5A medium. Following treatment, the cells
were washed with phosphate buffered saline (PBS) and then re-cultured in complete McCoy‟s 5A medium for 48 h. Subsequently, the cells were incubated with 0.5 mg/ml
of MTT in fresh complete McCoy‟s 5A medium for 4 h. The surviving cells converted MTT to formazan by forming a blue-purple color when dissolved in dimethyl sulfoxide.
The intensity of formazan was measured at 565 nm using a microplate reader (VERSAmax, Molecular Devices Inc., CA). The relative percentage of surviving cell was calculated by dividing the absorbance of treated cells by that of the control in each experiment.
2.5. Annexin V-PI assay
The level of apoptosis induced by ACP-93 was determined by Annexin V-propidium iodide (PI) analysis. The securin-wild type and securin-null HCT116 cells were treated with 0-32 μM ACP-93 for 24 h at 37°C. After treatment, the floating and adherent cells were collected and centrifuged at 1500 rpm for 5 min. Thereafter, the cell pellets were resuspended in 1× Annexin V binding buffer. Each sample was incubated
with fluorescein isothiocyanate (FITC)-conjugated-Annexin V and PI according to the manufacturer‟s instruction (BioVision, Mountain View, CA) for 5 min at room
temperature. Finally, the samples were analyzed immediately using flow cytometer (FACS Calibur, BD Biosciences, Heidelberg, Germany). For each measurement, 10,000 cells were analyzed. Dot plots and histograms were analyzed by CellQuest software (BD Biosciences). Annexin V-/PI- cells were viable. The cells showed Annexin V+/PI -and Annexin V+/PI+ , which indicated at early and late apoptosis, respectively.
2.6. Cell cycle analysis
The cell cycle progression after treatment with ACP-93 was measured by flow cytometry. The cells were plated at a density of 1 × 106 cells per 60-mm Petri dish in complete medium for 16-20 h, and then treated with 0-32 μM ACP-93 for 24 h at 37°C.
At the end of treatment, the cells were collected and fixed with ice-cold 70% ethanol overnight at -20°C. Thereafter, the cell pellets were treated with 4 μg/ml PI solution containing 1% Triton X-100 and 50 μg/ml RNase for 30 min. To avoid cell aggregation, the cell solutions were filtrated through nylon membrane (Becton-Dickinson, San Jose, CA). Subsequently, the samples were analyzed by flow cytometry. For each measurement, 10,000 cells were analyzed for DNA content, and the percentage of cell cycle phases were quantified by ModFit LT software (Ver. 2.0, Becton-Dickinson).
2.7. Western blotting
At the end of treatment, the cells were lysed in the ice-cold whole cell extract buffer (pH 7.6) containing the protease inhibitors. The buffer containing 0.5 mM DTT, 0.2 mM EDTA, 20 mM HEPES, 2.5 mM MgCl2, 75mM NaCl, 0.1 mM Na3VO4, 50 mM NaF, 0.1% Triton X-100, 1 mg/ml aprotinin, 0.5 mg/ml leupeptin, and 100 mg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride. The lysate was vibrated for 30 min at 4°C and centrifuged at 10,000 rpm for 10 min. The protein concentrations were determined by the BCA protein assay kit (Pierce, Rockford, IL). The total cellular protein extracts were prepared. Equal amounts of proteins were subjected to electrophoresis using 10 to 12% sodium dodecyl sulfate-polyacrylamide gels and electrophoretic transfer of proteins onto polyvinylidene difluoride membranes. The membranes were blocked overnight at 4°C using blocking buffer (5% non-fat dried milk in solution containing 50 mM Tris/HCl (pH 8.0), 2 mM CaCl2, 80 mM sodium chloride, 0.05% Tween 20 and 0.02% sodium azide). The membranes were sequentially hybridized with specific primary antibody and followed with a horseradish peroxidase-conjugated secondary antibody. Thereafter, the protein bands were visualized on the X-ray film using the enhanced chemiluminescence detection system (PerkinElmer Life and Analytical Sciences, Boston, MA). To verify equal protein loading and transfer, actin was used as the protein loading control. The gel digitizing software, Un-Scan-It gel (Ver. 5.1, Silk
Scientific, Inc., Orem, UT), was used to analyze the intensity of bands on X-ray film.
2.8. ROS measurement
The ROS induction after treatment with ACP-93 was measured by flow cytometry.
The cells were plated at a density of 7 × 105 cells per 60-mm Petri dish in complete medium for 16-20 h, then treated with 0-32 μM ACP-93 for 24 h at 37°C. After
treatments, the cells were collected and resuspended in medium containing 5 μM 2‟,7‟-dichlorodihydro-fluorescein diacetate (H2DCF-DA). The compound is
deacetylated by intracellular esterases converted to nonfluorescent dichlorodihydrofluorescein that reacts with H2O2 to form dichlorofluorescein (DCF), which is an oxidized green fluorescent compound. Cellular ROS content was measured by incubating the cells with stain solution for 30 min at room temperature in darkness.
Finally, the collected cell pellets were resuspended in 1 ml ice-cold PBS and fluorescence was detected by flow cytometer (FACScan, Becton Dickinson, San Jose, CA). Data from 10,000 events per sample were collected and analyzed using the CellQuest software.
2.9. Xenografted human colon tumors in SCID and nude mice
Human colorectal carcinoma xenograft was developed in 4 week-old
CB17/Icr-Prkdcscid/Crl mice that were obtained from BioLASCO (BioLASCO Co., Ltd., Taipei, Taiwan). 5 week-old BALB/cAnN.Cg-Foxn1nu/CrlNarl mice were obtained from National Laboratory Animal Center (NLAC, Taiwan). After 1-2 weeks for environmental adaption, the mice were used for human colorectal cancer cell inoculation. For pre-treatment of ACP-93, the securin wild-type HCT116 cells were treated with or without 32 μM ACP-93 prior to inoculation into the flank of the severe combined immunodeficiency (SCID) mice (1 × 106 cells/mouse) by subcutaneous injection. The tumor size and the body weight of the mice were measured every four days. For post-treatment of ACP-93, solid HCT116 flank tumors were established by
subcutaneous injection of 1 × 106 cells. After the tumors were visible, they received a 100 μl intra-tumoral injection of control vehicle (corn oil) or 30 mg/kg of ACP-93 once
per four days during 12 days. The tumor size of the mice was measured by a digital caliper every four days and calculated by the following formula: (length) × (width)2 × 0.5. The tumors volumes were continuously measured until the mice were sacrificed.
2.10. Securin-wild type and securin-null colon tumor in xenograft nude mice
Human colorectal carcinoma xenograft was developed in 4 week-old CAnN.Cg-Foxn1nu/CrlBltw mice that were obtained from BioLASCO (BioLASCO Co., Ltd., Taipei, Taiwan). Solid securin-wild type and securin-null flank colon tumors were
established by subcutaneous injection of 2 × 106 cells. The tumor size and the body weight of the mice were recorded every four days until mice were sacrificed.
2.11. Statistical analysis
Each experiment was repeated at least three times. Data were analyzed using Student‟s t test or analysis of variance (a comparison of multiple groups), and a p value
of < 0.05 was considered statistically significant in each experiment.