2.1. Chemicals and reagents
Butein (B-178), Hoechst 33258, Cy3-labeled mouse anti--tubulin (c-4585),
propidium iodide, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and the Cy3-labeled mouse anti--tubulin were purchased from Sigma
Chemical (St. Louis, MO). Butein was dissolved in DMSO, and the concentration of
DMSO was < 1 % in the control and drug-containing medium.
2.2. Antibodies
Anti-CDC2, anti-phospho-CDC2 (Tyr15), anti-phospho-CDC2 (Thr14),
anti-phospho-CDC2 (Thr161), anti-phospho-histone H3 (Ser10), anti-phospho-p53
(Ser15), and anti-poly (ADP-ribose) polymerase (PARP) was purchased from Cell
Signaling Technology, Inc. (Beverly, MA, USA). Anti-caspase-3 antibody was
purchased from BioVision (BioVision, Inc., USA). Anti-cyclin B1 (Ab-2) antibody
was purchased from Oncogene Sciences (Cambridge, MA). Anti-actin (I-19) antibody,
goat anti-rabbit IgG horseradish peoxidase, and goat anti-mouse IgG horseradish
peoxidase were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Antibody Source Molecular Weight Brand Catalog
Actin mouse 42 Santa Cruz sc-1616
CDC2 rabbit 34 Oncogene PC-25
Cyclin B1 mouse 60 Oncogene CC03
Caspase-3 mouse 32 Biovision 3004-100
PARP rabbit 17 Cell Signaling Tech #9542
Phospho-CDC2 (Thr161) rabbit 34 Cell Signaling Tech #9114
Phospho-CDC2 (Tyr15) rabbit 34 Cell Signaling Tech #4539
Phospho-CDC2 (Thr14) rabbit 34 Cell Signaling Tech #2543
Phospho-p53 (Ser15) rabbit 53 Cell Signaling Tech #9284
Phospho-histone H3 (Ser10) rabbit 17 Cell Signaling Tech #9701
Securin mouse 22 Abcam ab3305
Anti-rabbit IgG-HRP goat secondary antibody Santa Cruz sc-2004 Anti-mouse IgG-HRP goat secondary antibody Santa Cruz sc-2005 Anti-rabbit IgG-Hylite 488 goat secondary antibody Jackson 115-485-003
2.3. Cell culture
The wild type, securin (-/-), and p53 (-/-) HCT116 colorectal carcinoma cell lines
were kindly provided by Dr. B. Vogelstein of Johns Hopkins University (Baltimore, MD). These cell lines were cultured in complete McCoy‟s 5A medium (Sigma
Chemical) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/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 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 treated with various concentrations of butein for 24 h,
and then the cells were washed with phosphate-buffered saline (PBS) and were
replaced fresh complete McCoy‟s 5A medium for cultured 2 days. 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 cell viability was calculated by dividing the absorbance of
treated cells by that of the control in each experiment.
2.5. Time-lapse observation of cell death
HCT116 colon cancer cells were plated at a density of 2 × 105 cells per 35-mm
Petri dish in complete medium for 16-20 h. Then the cells were treated with or
without 40 μM butein by time-lapse observation under an optical phase contrast
microscope with an incubator system (OLYMPUS IX71, Japan). The pictures were
edited by DP manager software (Ver. 3.3.1, OLYMPUS)
2.6. Cell cycle analysis
The cell cycle progression after treatment with butein was measured by flow
cytometer. The cells were plated at a density of 1 × 106 cells per 60-mm Petri dish in
complete medium for 16-20 h. After treatment, the cells were collected and fixed with
ice-cold 70% ethanol overnight at -20 °C. After centrifugation, the cell pellets were
treated with 4 μg/ml PI solution containing 1% Triton X-100 and 100 μg/ml RNase at
37 °C for 30 min. After re-centrifugation, the cells resuspended in 1 ml ice-cold PBS.
To avoid cell aggregation, the cell solutions were filtrated through nylon mesh
membrane. Subsequently, the samples were analyzed by CellQuest software in flow
cytometer (BD Biosciences, San Jose, CA). A minimum of ten thousand cells was
analyzed for DNA content, and the percentage of cell cycle phases was quantified by
ModFit LT software (Ver. 2.0, Becton-Dickinson).
2.7. Annexin V and PI assays
The cells were plated at a density of 7 × 105 cells per 60-mm Petri dish in
complete medium for 16-20 h. Thereafter, the cells were treated with 0-40 μM butein
for 24 h. Apoptotic cells was performed using an annexin-V-FITC Apoptosis Detection Kit (BioVision, Mountain View, CA) according to the manufacturer‟s
instructions. Then cells were collected and resuspended in 500 μl of binding buffer,
added 5 μl of annexin-V-fluorescein isothiocyanate (FITC) and 5 μl of propidium
iodide (PI). Finally, the samples were analyzed by flow cytometer using CellQuest
software (FACScan, Becton–Dickinson, San Jose, CA). The cells showed annexin V(+)/PI(−) and annexin V(+)/PI(+), which indicated at early and late apoptosis,
respectively.
2.8. Western blot
After the end of drug treatment, the cells were lysed in the ice-cold whole cell
extract buffer containing the protease inhibitors. The lysate was vibrated for 30 min at
4 ℃ and centrifuged at 10,000 rpm for 10 minutes. Protein concentration was
measured by BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of proteins
were subjected to electrophoresis using 12 % sodium dodecyl sulfate-polyacrylamide
gels. To verify equal protein loading and transfer, proteins were then transferred to
polyvinylidene difluoride membranes and the membranes were blocked overnight at 4
℃ 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 then incubated for 2 h at 25℃ with specific
primary antibody followed by anti-rabbit or anti-mouse immunoglobulin
G-horseradish peroxidase conjugated secondary antibodies. The membranes were
washed three times for 10 min with washing solution. Finally, the protein bands were
visualized on the X-ray film using the enhanced chemiluminescence detection system
(PerkinElmer Life and Analytical Sciences, Boston, MA). A gel-digitizing software,
Un-Scan-It gel (ver. 5.1; Silk Scientific, Inc.), was used to analyze the intensity of
bands on X-ray film by semi-quantification.
2.9. Immunofluorescence staining and confocal microscopy
The cells were cultured on coverslips, which were kept in 35-mm Petri dish at a
density of 5 × 105 per well for 16-20 h. After treatment with or without 40 μM butein
for 24 h, the cells were washed with PBS. Then fixation with 4% paraformaldehyde
solution overnight at 4 °C, the cells were washed three times with PBS, and
non-specific binding sites were blocked in PBS containing 10 % FBS and 0.3 %
Triton X-100 for 1 h at 37 °C. Thereafter, the cells were separately incubated with
rabbit anti-phospho-histone H3 (1:100) antibody in PBS containing 10 % FBS
overnight at 4°C, and washed three times with 0.3 % Triton X-100 in PBS. Then the
cells were incubated with anti-rabbit IgG-Hylite 488 (1:100) in PBS containing 10 %
FBS for 1 h at 37 °C, and washed three times with 0.3 % Triton X-100 in PBS. The
samples incubated with mouse anti-securin (1:100) antibody in PBS containing 10 %
FBS overnight at 4°C, and washed three times with 0.3 % Triton X-100 in PBS. Then
the cells were incubated with anti-mouse IgG-Cy3 (1:100) in PBS containing 10 %
FBS for 1 h at 37 °C. The β-tubulin and nuclei were stained with the Cy3-labeled
anti-β-tubulin and Hoechst 33258, respectively. After staining, the samples were
immediately examined under Olympus confocal microscope (Olympus, Tokyo,
Japan).
2.11. Mitotic index analysis
The cells were cultured on coverslips in a 35-mm Petri dish at a density of 5 ×
105 for 16-20 h. After treatment with or without 40 μM butein for 24 h, the cells were
carefully and gently washed with PBS (pH 7.4) and then fixed with 4%
paraformaldehyde solution in PBS for one hour at 37 ºC. The cells were incubated
with rabbit anti-phosphorylated histone H3 (Ser10) antibody. Then the cells were
incubated with goat anti-rabbit IgG-Hylite 488. The -tubulin was stained with the Cy3-labeled mouse anti--tubulin (1:50) for 30 min at 37 ºC. Finally, the nuclei were
stained with 2.5 g/mL Hoechst 33258 for 30 min. Mitotic index indicated the
percentage of mitotic cell number/total counted cells that was counted under a
fluorescence microscope in each treatment. The prophase, metaphase, anaphases, and
telophase in total mitotic phases were counted under fluorescence microscope.
2.12. Analysis of phospho-histone H3 by flow cytometer
After treatment with butein for 24 h, HCT116 cells were harvested and fixation
with 75% alcohol in -20℃ overnight. Subsequently, the samples were incubated with
10 % bovine serum albumin in PBS for 1 hour at 4 °C. After that, the samples were
incubated with rabbit anti-phospho-histone H3 antibody (1:100), and following
incubated with anti-rabbit IgG-Hylite 488 (1:100) for 2 hours at 4 °C in dark. At the
end of incubation, the cells were resuspended in 1 × PBS and immediately analyzed
by a flow cytometer (FACS Calibur, BD Biosciences). The fluorescence intensities of
phospho-histone H3 were quantified the green fluorescence using CellQuest software
(BD Biosciences).
2.13. Statistical analysis
Each experiment was repeated at least three times. Data from the population of cells treated with different conditions were analyzed using paired Student‟s t-test. In a
comparison of multiple groups, data were analyzed by one-way or two-way analysis of variance (ANOVA), and further post Tukey‟s tests using the statistic software of
GraphPad Prism 5 (GraphPad software, Inc. San Diego, CA). A p value of < 0.05 was
considered as statistically significant in each experiment.