2.1 Chemicals and reagents
Gefitinib was purchased from proteinkinase.de (Biaffin GmbH & Co KG, Kassel, Germany) and dissolved in DMSO. Gefitinib analogues were kindly provided by Dr. C.
Chen (National Dong-Hwa University, Hualien, Taiwan) and dissolved in DMSO. 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI), the Cy3-labeled mouse anti-β-tubulin (c-4585) and Hoechst 33258 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3,3’-dihexiloxadicarbocyanine (DiOC6) was purchased from Calbiochem (San Diego, CA, USA). BODIPY FL phallacidin (B-607) was purchased from Invitrogen (Carlsbad, CA, USA). A431 cell lysate (12-301) were purchased from MILLIPORE (Temecula, CA, USA).
2.2 Antibodies
Anti-phospho-EGFR (05-1128) and anti-EGFR (05-104) antibodies were purchased from MILLIPORE (Temecula, CA, USA). Anti-caspase-3 (3004-100) was purchased from BioVision Research Products (Mountain View, CA, USA). Anti-PARP (#9542) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-securin (ab-3305) was purchased from Abcam (Cambridgeshire, UK).
Anti-ATF3 (sc-188) and the FITC (fluorescein isothiocyanate)-labeled goat anti-mouse IgG (sc-2010) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The Cy3-labeled goat anti-rabbit IgG was purchased from Amersham Pharmacia Biotech (Little Chalfont Buckinghamshire, UK). Anti-actin (MAB1501) antibodies were purchased from CHEMICON International, Inc. (Temecula, CA, USA).
2.3 Cell lines and cell culture
RKO was a colorectal carcinoma cell line that expressed the wild type p53 proteins.
The A549 cell line was derived from lung carcinoma that contained the wild type p53.
BFTC905 cells were derived from bladder carcinomas of Chinese patients. The MCF7 cell line was derived from breast adenocarcinoma of 69 years adult female. A375 cells were derived from skin carcinomas of malignant melanoma. The securinwild type and -null HCT116 colorectal carcinoma cell lines were kindly provided by Dr. B. Vogelstein of Johns Hopkins University (Baltimore, MD). RKO and A375 cells were maintained in DMEM medium (Gibco, Life Technologies, Grand Island, NY). A549, BFTC905 and MCF7 cells were cultured in RPMI-1640 medium (Gibco, Life Technologies, Grand Island, NY, USA). The securin-wild type and -null HCT116 cells were cultured in McCoy’s 5A medium (Sigma Chemical). The complete medium was 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 overnight. Following gefitinib of its analogues treatment for 24 h, the cells were washed with phosphate-buffered saline (PBS) and were replaced fresh medium for cultured 2 days. Thereafter, the medium was replaced and the cells were incubated with 0.5 mg/ml of MTT in complete medium for 4 h. The surviving cells converted MTT to formazan that generates a blue-purple color when dissolved in dimethyl sulfoxide. The intensity of formazan was measured at 565 nm using a plate reader (Molecular Devices, VERSAmax). 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 Cell cycle analysis
The cell cycle progression after treatment with gefitinib 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 overnight. Then the cells were treated with 0-60 μM gefitinib for 24 h. At the end of 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 (in the dark). After re-centrifugation, the cells resuspended in 1 ml ice-cold PBS.
To avoid cell aggregation, the cell solutions were filtrated through nylon membrane (Becton-Dickinson, San Jose, CA). Subsequently, the samples were analyzed by flow cytometer. A minimum of ten thousand cells was analyzed for DNA content, and the percentage of cell cycle phases was quantified by a ModFit LT software (Ver. 2.0, Becton-Dickinson).
2.6 Annexin V and PI assay
The level of apoptosis was determined by annexin V-PI staining analysis. The annexin V-PI staining kit (BioVision, Mountain View, CA) was used to examine the cells by incubated with fluorescein isothiocyanate (FITC)-conjugated-annexin V and PI according to the manufacturer's instruction. The cells were cultured in 60-mm Petri dish at a density of 1 × 106 cells for overnight. After treatment with or without gefitinib for 24 h, the cells were washed with PBS. The cells were trypsinized and collected by centrifugation at 1500 rpm for 5 min. Thereafter, the cells were incubated with 500 μL of annexin V-PI labeling solution (containing 5 μL of annexin V-FITC and 5 μL of PI) at 25 °C in the dark for 5 min. 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.
2.7 Observation of living cell image
To examine the effect of gefitinib on cell death. After the cells were plated at a density of 2 × 105 cells/p35 Petri dish for 24 h, the cells were exposed to 0 or 60 μM gefitinib. And then the cell morphology was observed under an imaging system of living cell observation in inverted microscope (OLYMPUS, IX71, Japan) for 24 h.
2.8 Mitochondrial membrane potential assay
Mitochondrial function was evaluated by the cells stained with the mitochondrial sensitive probe DiOC6. Lipophilic cation DiOC6 accumulated in the mitochondrial matrix driven by the electrochemical gradient. The cells were cultured in 60-mm Petri dish at a density of 1 × 106 cells for overnight. After treatment with or without gefitinib, the cells were washed with ice-cold PBS. The cells were trypsinized and collected by centrifugation. Then cell pellets were incubated with 50 nM DiOC6 in complete medium at 37 °C for 30 min (in the dark). Finally, the cell pellets were collected by centrifugation and resuspended in 1 ml ice-cold PBS and analyzed by flow cytometer (FACScan, Becton Dickinson, San Jose, CA).
2.9 Western blot
The cells were plated at a density of 6 × 106 cells per p100 Petri dish in complete medium for overnight. Then the cells were treated with 0-60 μM gefitinib for 24 h. At the end of treatment, the cells were lysed in the icecold cell extract buffer (pH 7.6) 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. The protease inhibitors including 1 μg/ml aprotinin, 0.5 μg/ml leupeptin, and 100 μg/ml 4-(2-aminoethyl) benzenesulfonyl fluoride were added to the cell suspension. The protein concentrations were determined by the BCA protein assay kit (Pierce, Rockford, IL). The total cellular protein extracts were prepared. And they were separated on 6-12% sodium dodecyl sulfate-polyacrylamide gels, and electrophoretic transfer of proteins onto polyvinylidene difluoride membranes. The membranes were sequentially hybridized with primary antibody and followed with a horseradish peroxidase-conjugated secondary antibody.
The protein bands were visualized on the X-ray film using the enhanced chemiluminescence detection system (PerkinElmer Life and Analytical Sciences, Boston, MA). Western analyses of various phospho-EGFR, total EGFR, caspsae-3, PARP, securin, ATF3 and actin were performed using specific antibodies. To verify equal protein loading and transfer, actin was used as the protein loading control. 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.
2.10 Immunofluorescence staining and confocal microscopy
To view the localization and expression of proteins after gefitinib treatment, the cells were subjected to immunofluorescence staining and confocal microscopy. The cells were cultured on coverslips, which were kept in 6-well plates at a density of 2 × 105 per well for overnight before treatment. After treatment with or without 40 μM gefitinib for 24 h, the cells were washed with isotonic PBS (pH 7.4). Then fixation with 4% paraformaldehyde solution for 1 h at 37 °C, the cells were washed three times with PBS. And non-specific binding sites were blocked in PBS containing 10% FBS and 0.25% Triton X-100 for 1 h at 37 °C, and washed three times with 0.25% Triton X-100 in PBS. Thereafter, the cells were incubated with mouse anti-securin (1:120) or rabbit anti-ATF3 (1:200) antibodies in PBS containing 10% FBS and 0.25% Triton X-100 overnight at 4 °C, and washed three times with 0.25% Triton X-100 in PBS. Then the cells were incubated with goat mouse FITC-labeled IgG (1:120) and goat anti-rabbit Cy3-labeled IgG (1:200) in PBS containing 10% FBS and 0.25% Triton X-100 for 2.5 h at 37 °C, and washed three times with 0.25% Triton X-100 in PBS. The β-tubulin, F-actin, and nuclei were stained with the Cy3-labeled anti-β-β-tubulin, BODIPY FL phallacidin and Hoechst 33258, respectively. Finally, the samples were stored in the dark until examined under a confocal microscope (OLYMPUS, Japan) that equipped with an UV laser (405 nm), an Ar laser (488 nm), and a HeNe laser (543 nm).
2.11 Transfection
The pCT-GFP2 and pCT-GFP-sec2 were employed for transfection using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's recommendations.
The cells were plated in 96-well plates at a density of 8 × 103 per well in complete medium for overnight, then were transfected with 50 μg/mL of control or securin-expressed vectors in 50 μL/well serum-free medium for 6 h at 37 °C in a CO2 incubator according to the manufacturer’s recommendations. Then, the equal amount medium with 20% fetal bovine serum was added without removing the transfection mixture, and incubation proceeded for an additional 24 h. After transfection, the cells were subjected to cytotoxicity analysis as described above.
2.12 Statistical analysis
Each experimentwas 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.