RDS 60 synthesis
RDS 60 (ethyl (5-((1H-pyrrol-1-yl)methyl)-1H-benzo[d]imidazol-2-yl)carbamate) (Fig. 1a) was synthesized as previously described [10]. However, we modified the first two reaction steps in order to improve the overall reaction yields. Indeed, we applied a reduction reaction according to Moore et al. [18] of the commercially available carboxylic acid into alcohol that was subsequently converted in the corresponding benzyl chloride (Fig. 1b) via an aliphatic nucleophilic substitution. The subsequent synthetic steps resemble the ones previously reported [10]. Therefore, we report the details of the first two synthetic steps since the other ones can be found in ref. 10. Melting point (°C), recrystallization solvent, yield (%), chromatographic system, IR, 1H NMR, formula, Mr and analyzed elements for derivative RDS 60 agreed with the ones previously described [10]. RDS 60 samples used for biological evaluation were 99% pure as determined by elemental analysis.
Synthesis of 1
A solution of AlCl3 (3.77 g; 28.28 mmol) in THF dry (4.6 mL) was added dropwise into a solution of 3,4-dinitrobenzoic acid (5 g; 23.57 mM) and NaBH4 (3.21 g; 84.85 mmol) in THF dry (46 mL) at 0 °C within 20 min. The reaction was stirred at 25 °C for 1 hour (h) and for 2 h to reflux and then poured into ice-cold water (115 mL). The pH was adjusted to 7 with 1 N HCl and the mixture was extracted with ethyl acetate (3 x 50 mL). The collected extracts were washed with brine (3 x 100 mL) and dried. Removal of the solvent furnished crude residue that was washed with ethyl acetate in order to discard the boron salt, yielding intermediate 1 (3.37 g, 72.1 %) as a yellow solid.
Synthesis of 2
To a solution of compound 1 (16.27 g, 81.85 mM) in CHCl3 (126 mL) was added PCl5 (6.63 g, 31.85 mM) portionwise at 0 °C within 15 min. The reaction mixture was stirred at 0 °C for 15 min and at 25 °C for 2.5 h and then poured into ice-cold water. The organic layer was washed with 5% w/v Na2CO3 (3 x 100 mL) and with NaClss (3 x 100 mL) and dried. Removal of the solvent furnished the crude compound 2 that underwent to the following step without further purification.
Cell cultures and treatment
Two HNSCC cell lines were utilized: tongue carcinoma CAL27 and pharynx carcinoma FaDu (ATCC, USA), and one human dermal fibroblast primary culture HF [19, 20]. Cells were grown in RPMI 1640 medium with 10% fetal calf serum (FCS), 2 mM glutamine and 50 U/mL penicillin-streptomycin (Sigma-Aldrich). The compound RDS 60 was solubilized in dimethylsulfoxide (DMSO) (Sigma) for a 10 mM stock solution and utilized to final concentrations from 100 nM to 10 μM for 24 and 48 h. Control cells were treated with equivalent amounts of DMSO in every experiment.
Cytotoxicity assay
To determine cytotoxicity, a sulforhodamine-B colorimetric assay was performed: 5x103 cells were plated in 96-well plates, grown for 24 h and then treated with 100 nM, 1, 2.5, 5, 10 μM RDS 60 for 24 and 48 h. Cells were then fixed with 50% trichloroacetic acid for 1 h to 4 °C and stained for 30 min to room temperature (RT) with 0.4% sulforhodamine-B in 1% acetic acid. Excess dye was removed by washing four times with 1% acetic acid. Protein-bound dye was dissolved in 10 mM Tris pH 10 and optical density was determined at 510 nm using a microplate reader.
Flow cytometry analysis
Cells were seeded in 60-mm plates, grown for 24 h and then treated with 1 μM RDS 60 or equivalent amounts of DMSO for 24 h. The cells were harvested by trypsinization, washed twice with cold PBS, fixed in 70% ethanol at 4°C overnight. Cells were rinsed twice with PBS and incubated with 50 µL of RNAse (100 µg/mL, Sigma) to ensure that only DNA was stained, and 200 µL of Propidium Iodide (PI) (50 µg/mL, Sigma). Cell cycle analysis was performed using a FACS CantoII equipped with 488nm laser and DIVA Software (BD Biosciences). The cells were first gated using a forward vs. side scatter (FSC vs. SSC) strategy, and upon 488 nm laser excitation, PI fluorescence was then detected above 580 nm. Data were analyzed using Flow Jo software (Flow Jo LLC, OR, USA).
Immunofluorescence
Cells were grown on Labteck chamber slides (Nunc) for 24 h and then treated with 1μM RDS 60 or with DMSO for 24 h. Cells were washed with PBS with Ca/Mg (washing buffer) and fixed with 4% buffered paraformaldehyde (Sigma Aldrich) for 20 min at 4 °C, then permeabilized with PBS, 5% FCS, 0.5% TritonX100 for 30 min at RT and incubated for 1 h to RT with the primary monoclonal antibody to beta-tubulin (1:100 diluted; Immunological Sciences). Alternatively, cells were permeabilized with 0.1 % TritonX100 for 10 min to RT, incubated with 3% BSA for 1 h to RT and incubated with the primary rabbit polyclonal antibody to cyclin B1 in 0.1% BSA (1:200 diluted; Elabscience, USA) overnight at 4°C. Cells were washed twice with washing buffer and incubated with the secondary anti-mouse or anti-rabbit antibody FITC conjugated (1:400 diluted; Molecular Probes) for 1 h to RT. Cells were washed twice with washing buffer and DNA was stained with Hoechst for 15 min to RT. The slide was mounted with ProLong-Antifade (Life Technologies) and analyzed by a fluorescence microscope (Olympus BX52). Image acquisition and processing were conducted by IAS 2000 software.
Western blot analysis
Cells were seeded in 100-mm plates, grown for 24 h and treated with 2.5 μM RDS 60 or with equivalent amounts of DMSO for 24 h. Cells were then scraped in lysis buffer (1% Triton, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 50 mM Tris HCl pH 7.4, 2 mM EDTA) with protease inhibitor cocktail (Roche Applied Science, Germany) for 30 min at 4 °C. Lysates were centrifuged at 16,000xg for 15 min at 4 °C and the supernatant was collected. Protein concentration was evaluated using Protein Concentration Assay (Bio-Rad Laboratories, CA, USA). Protein lysates (50-100 μg) were separated by molecular weight with 10, 12 or 14% SDS-PAGE and then transferred onto nitrocellulose membranes. Membranes were blocked for 1 h to RT in 5% nonfat dry milk and incubated with primary antibodies opportunely diluted in PBS-Tween overnight at 4°C, washed in Tris-buffered saline with 0.1% Tween-20 and incubated with horseradish-peroxidase-conjugated-anti-mouse/rabbit-IgG (1:5000; Sigma-Aldrich, MO, USA) for 1 h to RT. Filters were then developed using enhanced chemiluminescence (Super Signal West Pico Chemiluminescence Substrate; Thermo Fisher Scientific, USA) using Kodak X-Omat films (Kodak, USA). Primary antibodies were: mouse anti-poly (ADP-ribose)-polymerase (PARP-1) (diluted 1:500; Santa Cruz Biotechnology, TX, USA); mouse anti-cleaved-caspase 8 (diluted 1:500; Cell Signaling Technology, MA, USA); mouse anti-caspase 9 (diluted 1:500; Cell Signaling Technology); mouse anti-B-cell-lymphoma-2 (Bcl-2) (diluted 1:200; Santa Cruz Biotechnology); rabbit anti-Bcl-2-associated-X-protein (Bax) (diluted 1:250; Santa Cruz Biotechnology); rabbit anti E-cadherin (1:1000 diluted; GeneTex); rabbit anti N-cadherin (1:1000 diluted; GeneTex); rabbit anti-tubulin (diluted 1:4000; Immunological Sciences). Experiments were performed in triplicate, the bands from the blots were quantified using ImageJ v.1.48 software (National Institutes of Health, Bethesda, MD, USA), the mean values were calculated and expressed as densitometric units (DU).
Invasion assay
Invasion assay was performed with Matrigel Invasion Chambers (Corning) consisting of inserts with 8 μm pore membrane pretreated with Matrigel: 5x105/mL cells were plated in serum-free medium plus DMSO or in serum-free medium plus 1 μM RDS 60 in the insert chamber, the lower chamber contained complete medium with FCS. After 18 h of culture the inserts were washed with PBS with Ca/Mg and fixed by 100% methanol for 20 min at 4 °C, washed twice with PBS with Ca/Mg and stained for 20 min at RT with haematoxylin. The inserts were then mounted on slides and the cells that migrated through the filter pores to the lower side of the membrane were counted by an optical microscope (Olympus BX52). Imagine acquisition and processing were conducted by IAS 2000 software.
Statistical analysis and graphic programs
All results were analyzed using one-way analysis of variance, and significance was evaluated using Tukey’s honest significant difference post hoc test. All figures were created using Adobe Photoshop CS5 and all graphs were produced and statistical analyses conducted using Graph Pad Prism 5.0.