2.1 Chemicals and antibodies
PT-262 was synthesized and kindly provided by our collaborator Dr. Chinpiao Chen (National Dong Hwa University, Hualien, Taiwan).Hoechst 33258, propidium iodide and 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical (St. Louis, MO). BODIPY FL phallacidin (B-607) was purchased from Invitrogen (Carlsbad, CA). Anti-p38, anti-survivin, anti-E-cadherin and anti-ERK-2 were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Anti-XIAP and anti-phospho-p38 (Thr180/Tyr182) were purchased from Cell Signaling Technology, Inc. (Beverly MA, USA). Anti-caspase-3 was purchased from BioVision, Inc. (San Francisco, CA). Anti-PARP was purchased from Clontech Laboratories, Inc. (Mountain view, CA). Anti-GAPDH was purchased from Novus Biologicals Inc. (Littleton, CO). Anti-γ-H2AX was purchased from BD Bioscience (Franklin Lakes, NJ). Anti-β-catenin was purchased from Abcam Inc. (San Francisco, CA).
2.2 Cell culture
The A549 cell line (ATCC, #CCL-185) was derived from human lung carcinoma.
A549 cells were cultured in complete RPMI-1640 medium (Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and L-glutamine (0.03 %, w/v). The cells were maintained at 37 °C and 5 % CO2 in a humidified incubator (310/Thermo, Forma Scientific, Inc., Marietta, OH).
2.3 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 treated with various concentrations of PT-262 for 24 h in complete RPMI-1640 medium. After treatment, the cells were washed once with phosphate buffered saline (PBS) and were re-cultured in complete RPMI-1640 medium for two days. Subsequently, the cells were incubated with 0.5 mg/ml of MTT in fresh complete RPMI-1640 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 cells was calculated by dividing the absorbance of treated cells by that of the control in each experiment.
2.4 Annexin V-propidium iodide analysis
For detection of apoptotic events, floating and adherent cells were collected.
A549 cells were treated with 0-30 μM PT-262 for 24 h at 37 ℃. After PT-262 treatment, the cells were centrifuged and then the cell pellets were suspended in 1 × Annexin V binding buffer. Samples were incubated with fluorescein isothiocyanate (FITC)-conjugated-Annexin V and propidium iodide (PI) according to the manufacturer’s instruction (BioVision, Mountain View, CA) for 5 min at room temperature. Then cells were analyzed immediately using a 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). The cells showed Annexin V positive and PI negative indicate at early apoptosis; while the cells showed Annexin V positive and PI positive indicate at late apoptosis. Annexin V and PI negative cells were termed viable.
2.5 Western blotting
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 30 min. Protein concentration was measured by BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of proteins (40–60 μg/well) were subjected to electrophoresis using 10 to 12 % sodium dodecyl sulfate -polyacrylamide gels. To verify equal protein loading and transfer, ERK-2 or GAPDH were used as the protein loading control. 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., Orem, UT), was used to quantify the
intensity of each band on the X-ray film.
2.6 RT-PCR
Cells were plated at a density of 2 × 106 cells per 60-mm petri dish in culture medium. Total cellular RNA was purified by Trizol reagent (Invitrogen) according to the manufacturer’s protocol. RNA concentrations were determined by spectrophotometry. cDNAs were synthesized by SuperScriptTM III reverse transcriptase with oligo (dT)12-18 primer (Invitrogen) Each reverse transcript was amplified with GAPDH as an internal control. The following primer pairs were used for amplification, E-cadherin forward primer: 5’-CAGTCAAAAGGCCTCTACGG-3’
and E-cadherin reverse primer: 5’-GTGTATGTGGCAATGCGTTC-3’; GAPDH forward primer: 5’-CGGAGTCAACGGATTTGGTC GTAT-3’ and GAPDH reverse primer: 5’-AGCCTTCTCCATGGTGGTGAAGAC-3’. RT-PCR was performed by a DNA thermal cycler (Mastercycler gradient, Hamburg, Germany). The initial denaturation was performed at 94 °C for 2 min, followed by 30 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 40 s; and 72 °C for 5 min. The PCR products were visualized on 1.2 % agarose gels with ethidium bromide staining under UV transillumination, and photograph was taken by a camera (DH27-S3, Medclub,
Taoyuan, Taiwan).
2.7 Immunofluorescence staining and confocal microscopy
To view the protein localization and expression of β-catenin after PT-262 treatment, the cells were subjected to immunofluorescence staining and confocal microscopy. After fixation with 4 % paraformaldehyde solution, 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. Thereafter, the cells were separately incubated with rabbit anti-β-catenin (1:200) 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 goat anti-rabbit Cy3 (1:200) in PBS containing 10
% FBS for 1 h at 37 °C. F-actin and nuclei were stained with BODIPY FL phallacidin and Hoechst 33258, respectively. The samples were examined under a confocal microscope Fluoview 300 (Olympus, Tokyo, Japan).
2.8 The cell surface expression of E-cadherin analyzed by flow cytometry
After treatment with PT-262 for 24 h, A549 cells were harvested and incubated
with 10 % bovine serum albumin in PBS for 30 min at 4 ℃. Subsequently, the samples were incubated with mouse anti-E-cadherin antibody (1:100), and followed incubated with a FITC-coupled goat anti-mouse antibody (1:200) for 30 min at 4 °C.
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 E-cadherin were quantified the green fluorescence using CellQuest software (BD Biosciences).
2.9 Anchorage-independent assay
To evaluate the effect of PT-262 on colony forming ability of lung cancer cells in soft agar, anchorage-independent assay was analyzed in this study. A549 cells were treated with or without 20-30 μM PT-262 for 24 h. After treatment, the cells were then seeded 2500 cells/well in 1 ml of 0.33 % agarose in 1 × RPMI medium with 1 ml of 0.5 % agarose in 1 × RPMI medium as the based layer . The cultures were maintained in a 37 °C, 5 % CO2 incubator for 20 days. Cells were fed 1-2 times per week with fresh culture medium. Finally, the colonies and numbers were counted under a phase contrast microscope. Colony numbers (more than 10 cells existing in a colony) were calculated from 10 random fields of each treatment.
2.10 Animal studies
CB17/Icr-Prkdcscid/CrlBltw mice (4-week-old male) were purchased from BioLASCO (Taipei, Taiwan). After 1-2 weeks for environmental adaption, the mice were used for human lung cancer cell inoculation. Two treatment methods were executed as followed. For pre-treatment of PT-262, A549 cells were treated with or without 20-30 μM PT-262 prior to inoculation into flanks of the severe combined immunodeficiency (SCID) mice (1 × 106 cells / mouse). The tumor size and the body weight of the mice were recorded every four days since then until day 64. For post-treatment of PT-262, solid A549 flank tumors were established by subcutaneous injection of 2 × 106 cells. When tumors reached volumes of 100 mm3, they received a 100 μL intra-tumoral injection of Tween 80/PBS (vehicle) or 5 mg/kg of PT-262.
The injections administrated every four days starting at day 0 and ending on day 8.
The tumor volume was continuously measured until the mice were sacrificed due to excessive tumor size or serious trauma was observed at the surface of the tumors.
Tumor measurement was performed using calipers, and tumor size was calculated using the ellipsoid formula 1/2 (LW2), where L represents length (the longest part of the tumor) and W represents width (the widest part).
2.11 Statistical analysis
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.