The synthetic compound, EMMQ MW 298.4, the structure shown in Fig 1A) [44], was purified to more than 98% purity and dissolved in DMSO at 10 mM for storage. Three cell lines, H460 (HTB177TM), H1299 (CRL5803TM) and A549 (CCL185TM) of human NSCLC cells and
MRC-5 of human Lung fibroblast cells (CCL-171 TM) were acquired from ATCC and maintained in DMEM and cultured with L-glutamine, sodium pyruvate, and supplied with 10% heat-inactivated FBS under 5% CO2 at 37 °C. The selected stable H1299 clones transfected with
cytomegalovirus promoter-driven pcDNA-p53 encoding full-length wild type p53 (H1299/p53) or mutant p53R267P (H1299/p53R267P) were maintained in 10 % serum-supplemented DMEM. Short hairpin RNA targeting p53 and scrambled non-specificity was acquired from the National RNAi Platform, Academia Sinica, Taipei, Taiwan.
2. Cell viability assay
The cell cytotoxicity was measured as previously described [45] . Briefly, cells were cultured at 1.5×103 cells per well in 96-well plates.
The attached cells supplemented with minimal amounts of FBS were treated with different concentration of cisplatin (Sigma), EMMQ or vehicle control DMSO at 37°C for 48 h. Cells were then kept in 10 μL of MTT (5 mg/mL) or cisplatin with medium at 37 °C for 4 h. MTT was then removed, added with 100 μL DMSO into each well and the
absorption measured by measuring the absorbance at 570 nm wavelength
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with a 96-well microplate reader (Thermo Fisher Scientific, USA).
3. Colony forming assay
Cells were seeded at 50 cells per well in a 6-well plates for 16 h to allow attachment and then added with EMMQ at different concentrations or vehicle control for 48 h. After replacing with fresh media and growing in 3 mL medium for 14 days, the colonies were fixed before staining with 0.5% methylene blue in ethanol for 4 h. The size and number of stained colonies with more than 50 cells were counted under inverted phase contrast microscope. Colony formation was calculated as a percentage to untreated control cultures.
4. Comet assay
Conventional slides were covered with a layer of 70 μL 0.5 % normal agarose and 0.5% low melting point agarose (GIBCO-BRL). A volume of 70 μL of low melting point agarose (0.5%, w/v) (GIBCO-BRL) was mixed with approximately 2 × 104 cells suspended in 15 μL of media.
The mixture was then layered onto the slides, and immediately overlaid with coverslips. After agarose solidification at 25 °C for 30 min, the coverslips were removed and the slides immersed 60 min at 4 °C in fresh lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10 and 1% Triton X-100). The slides were soaked in the alkaline buffer (300 mM NaOH and 1 mM Na2EDTA at pH 13) on the ice bath for 20 min and electrophoresed (30 V and 300 mA) for 20 min. Slides were then
transferred to the neutralization buffer (0.4 M Tris-HCl at pH 7.5) on ice
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bath for 15 min. Finally, slides were dried in methanol for 5 min and stained with 50 μL of PI (4 µg/mL). The tails were observed under a fluorescence microscope (Nikon, Japan) and quantified by using CometScore™ software (Tritek Corp, Sumerduck, VA).
5. Determination of apoptosis
Double staining with Annexin V-FITC and PI
Cells were seeded at 2 ×105 cells per well in 12-well plates and allowed to attach overnight. Cells were treated by various concentrations of EMMQ incubated at 37 °C for 48 h. DMSO containing medium served as the vehicle control. The cells were trypsinized and stained with1 μL annexin-V/FITC (20 µg/mL, BD Bioscience) and 1 μL of PI (50 µg/mL) at room temperature for 30 min in the dark. The early and late phase of apoptosis was measured by annexin V-FITC/PI Apoptosis Detection Kit (BD Bioscience). The flow cytometer FACS CaliburTM (BD Bioscience) was used for analysis and the data were analyzed using the FlowJo
software (Tree Star, Inc.).
Cell-cycle distribution
Cell cycle distribution was determined by suspending cells in 70 % ethanol and kept at -20 °C for 24 h prior to addition of 10 µg/mL of PI (Sigma, St. Louis, MO) and 10 µg/mL of RNaseA (ICN Pharmaceutical;
Costa Mesa, CA) in PBS (UniRegion Bio-tech, Taiwan) for 30 min. The data as acquired by flow cytometry were analyzed with software FlowJo (Tree Star, Inc.).
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6. Determination of ΔΨm
ΔΨm was determined using MitoPT™ JC-1 Assay Kit
(ImmunoChemistry Technologies, Bloomington, USA). Briefly, the seeded cells were cultured in 2% serum-supplemented DMEM containing different concentration of EMMQ or vehicle control and incubated at 37
°C for the time points as indicated. The collected cells were washed with 1× assay buffer. After centrifugation at 1,000 rpm for 5 min, cell pellets were stained with 250 μL mixture containing 5 μL of JC-1 with 995 μL 1× assay buffer for 25 min at 37 °C. The residual JC-1 was removed by centrifugation at 1,000 rpm for 5 min and the pellet mixed with 1× assay buffer. JC-1 fluorescence was measured to assess the emission shift from green (530 nm) to red (590 nm) using 488 nm excitation wavelength.
Data were given as the relative ratio of green to red fluorescence intensities, indicating the level of depolarization of the mitochondrial membrane potential. The data as determined by FACS CaliburTM were quantified and the expressed as the percentage of mitochondrial
membrane potential drop relative to those of untreated cells.
7. Release of cytochrome c release
The harvested cells after treatment were treated with 100 μL
digitonin (50 µg/mL PBS, 100 mM potassium iodide and 1 mM EDTA) for 5 min on ice until more than 95 % of cells permeabilized. Cells were then fixed and stained with 3.7 % formaldehyde and DAPI (1:3,000) (Sigma, USA) in PBS for 20 min at room temperature, washed thrice in
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PBS, and incubated in blocking buffer (3% bovine serum albumin (Themo, USA) and 0.05 % saponin in PBS) for 1 h. The cells were incubated overnight at 4°C with anti-cytochrome c mouse monoclonal antibody (BD PharMingen) that was diluted to 1:200 in blocking buffer, washed thrice, and incubated for 1 h at room temperature with
TRITC-conjugated goat anti-mouse (Santa Cruz) in blocking buffer. The cells were then counterstained with Mitotracker Green (Invitrogen Life Technologies). The samples were detected using a Leica TCS SP5 Confocal Spectral Microscope.
8. Western blot analysis
The western blot analysis was determined by electrophoresis of the protein contents of cell lysates were collected and the concentrations quantitated using BCA assay (Pierce Biotechnology, Rockford, IL). A total of 20 µg of protein were resolved by electrophoresis through
SDS-PAGE gel was transferred to nitrocellulose filters, blocked with 5%
of Skim Milk (BD, Mansfield, MA) and incubated with primary and secondary antibodies. The emitted chemiluminescence signals were visualized by ECL detection kit (Millipore).
9. Transfection with p53 shRNA
A549 and H460 NSCLC cancer cells were seeded in 60-mm dishes at 5 × 105 cells/dish, incubated overnight, and then transfected with p53 shRNA using Lipofectamine 3000 transfection reagent (Invitrogen, USA) according to the manufacturer’s protocol. After a 24 h transfection period,
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cells were treated with EMMQ for 48 h. Cell lysates were collected for western blot analysis.
10. Xenograft tumor evaluation
The athymic nu/nu female mice (BALB/c) of 3-4 week of age were obtained from the National Laboratory Animal Center (Tainan, Taiwan) and housed under pathogen-free conditions with a 12 h light/12 h dark schedule in the Animal Resource Facility at the animal center (Kaohsiung Medical University, Kaohsiung, Taiwan) in accordance with the
Institutional Animal Care and Use Committee guidelines. The animal study was performed according to protocols of the Institutional Animal Guidance. A total of 1×106 cells were suspended in 100 μL of PBS and were injected into dorsal legs hypodermic area of nude mice with a total of 6 mice in each group. The EMMQ was dissolved in DMSO and the mixture of PBS and Matrigel™ Basement Membrane Matrix (BD Biosciences, San Jose, CA) (4:1) before EMMQ treatment. Once the tumors reached 50 mm3, the mice were injected subcutaneous with 200 μL of EMMQ (at a dosage of 1 mg/kg/mouse) or vehicle control twice a week for four consecutive weeks. The size of each tumor was measured at each time point before EMMQ administration. The tumor volumes were calculated according to the formula: 1/2 × (length×width2). After 35 days of drug treatment, mice were sacrificed by CO2 inhalation and death was confirmed by cervical dislocation. The tumor samples were dissected were measured and lysed later for protein analysis. One-way ANOVA test was used for statistical comparisons between different groups.
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11. Cell migration assay
Migration was determined using the wound healing assays by A549, H1299 and H460 cells, respectively. Cells were seeded at 2 ×105 cells per well into 12-well plates for 16 h to allow cell attachment. Each NSCLC cells were treated different concentrations of EMMQ or solvent control DMSO and scraped with a 200 µL tip (time 0). Before imaging,
suspended cells were washed off. The distance of migrating cells was measured from images at 48 h after EMMQ treatment. The results of wound healing assay were normalized as ratio of wound repaired area to the non-treated control set to 100%.Wound closure was evaluated and photographed at 48 h with an inverted microscope (Nikon, Japan).
12. Gelatin zymography
A549 cells were starved for 24 h with serum-free medium.
Subsequently, cells in media containing 0.5% FBS were treated with EMMQ for different time periods and concentrations, and thereafter, the supernatants were collected. The samples were analyzed with gelatin zymography (0.1% w/v) to assess the enzymatic activity of MMPs by using gelatin as the substrate. Each lane was loaded with a total protein concentration of 3 µg and subjected to SDS-PAGE electrophoresis (30%
acrylamide, 10% SDS, 10% APS, TEMED, 1.5 M Tris (pH 8.8), 1.0 M Tris (pH 6.8)) at 48 °C. Gels were washed twice in 50 mM Tris (pH 7.4) containing 2.5% (v/v) Triton X-100 for 1 h, followed by repeated 10-min rinses in 50 mM Tris (pH 7.4). Gels were then incubated overnight in 50
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mM Tris (pH 7.5) containing 10 mM CaCl2, 0.15 M NaCl, 0.1% (v/v) Triton X-100, and 1% NaN3 at 37 °C with gentle shaking overnight. After incubation, gels were stained with 0.25% Coomassie brilliant blue and destained in 7.5% acetic acid with 20% methanol. Matrix
metalloproteinases in the loaded supernatants leads to the gelatinase bands appearing white on a blue background.
13. Statistical analysis
Experiments were performed 3 times. The differences between the treated and control cells were analyzed using the Student's t-test between two groups. The data were expressed as mean values ± SD of three independent experiments and p<0.05 was considered significant.
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