Reagents and chemicals
Colchicine (Cat#C9754), Nocodazole (Cat#M1404) and Paclitaxel (Cat#T1912) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Doxorubicin HCI (Doxorubicin, Adriamycin®), (Cas#25316-40-9) was obtained from Pfizer, Inc (New York City, New York, USA). Stock solutions of selected tested compound (MBIC) were maintained in dimethyl sulfoxide (DMSO), protected from light and stored at −20 °C for experimental purposes.
Test material
The synthesis of Methyl 2-(5-fluoro-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylate (2e) has been described previously [18] and is also mentioned below. MBIC was kindly supplied by Professor Dr. Piyush Trivedi, School of Pharmaceutical Sciences, Rajiv Gandhi Technical University, India. The chemical structure of MBIC is shown in Fig. 1a.
Procedure for the synthesis of Methyl 2-(5-fluoro-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylate (MBIC)
To a solution of the 5-fluoro-2-hydroxybenzaldehyde (2 mmol) in 6.5 ml of N,N-dimethyl acetamide was added 3,4 methyl 3,4-diaminobenzoate (2 mmol) and Na2S2O5 (2.4 mmol). The mixture was heated to 100 °C for 8 h until TLC confirms the completion of the reaction. The reaction mixture was then cooled, diluted with ethyl acetate (5 × 25 ml), dried (MgSO4), and concentrated in vacuo. The solid obtained was collected on a sintered-glass filter and washed with dichloromethane (3x) to provide the desired compound.
Seventy two percent yield; M.P. 239 °C; IR (KBr) ν (cm−1): 3333 (–NH), 3057 (Ar–C–H), 1722 (–C = O), 1255 (–C–N), 1213 (–CO–O) 1535 (Ar C–C); 1H NMR (400 MHz, DMSO-d6): δ (ppm) 13.12 (br s, 1H, NH), 8.22 (s, 1H, Ar H), 7.82–7.88 (m, 2H, Ar H), 7.67(s, 1H, Ar H), 6.96–7.12 (m, 2H, Ar H), 3.85 (s, 3H, CH3); LC–MS analysis (M - H)−: 285.0 (calculated 286.08); Elemental Analysis: Calcd. (Found) (%) for C15H11FN2O3: C 62.94 (62.90), H 3.87 (3.89), N 9.79 (9.80) [18].
Cell culture
The human cervical cancer cell-line, HeLa (CCL-2.2; ATCC, Manassas, VA), HCT-116 (CCL-247; ATCC, Manassas, VA), A549 (CCL-185; ATCC, Manassas, VA), HepG-2 (HB-8065; ATCC, Manassas, VA) and WRL-68 (CL-48; ATCC, Manassas, VA), were obtained from American Type Culture Collection (ATCC). HeLa, A549, HepG2 and WRL68 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies, Inc, Rockville, MD). HCT-116 was obtained in McCoy’s 5a Medium Modified (Life Technologies, Inc, Rockville, MD) supplemented with 10 % fetal bovine serum and 1 % penicillin/streptomycin. Cells were incubated at 37 °C in a humidified cell culture 5 % CO2 incubator. Cells were passaged until reaching 70-80-% confluency for experimental purposes.
Cytotoxicity assay
Cytotoxicity of MBIC was estimated by MTT assay. Briefly, 1 × 104 cells/well were seeded in a 96-well plate and incubated at 37 °C in 5 % CO2. After 24 h, cells were treated with different concentrations (0.02, 0.04, 0.1, 0.2, 0.4, 0.8, 1.5, 3, 6, 12, 25, 50 μM) of MBIC. 0.01 % DMSO was used as vehicle control. Cells were incubated for 24 and 48 h at 37 °C. Subsequently, 50 μl/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 2 mg/ml) was added and incubated for 2 h. After the incubation, the media was discarded and 100 μl DMSO was replaced into each well to dissolve the formazan crystal. The colorimetric assay was quantified at 570 nm wavelength using Chameleon V microplate reader (Hidex, Turku, Finland). The anti-proliferation activity of MBIC was expressed as an IC50 value. The percentage of cell viability was calculated as described previously [19].
Apoptosis analysis
Apoptosis was detected using BD Pharmingen Annexin V-FITC Apoptosis Detection Kit as per manufacturer’s protocol. Briefly, cells were seeded in 25 cm2 culture flasks (10 × 105 cells/flask) and were treated in 3 different MBIC concentrations for 24 and 48 h. Later, treated cells were harvested and washed with 1 ml of 1X Phosphate Buffered-Saline (PBS) before adding 200 μl 1X annexin-binding buffer. 10 μl of annexin V-FITC and 10 μl of PI were added to each 200 μl of cell suspension. Cells were then incubated in total darkness for 15 min at room temperature. 500 μl of 1X annexin-binding buffer was added just before the flow cytometry analysis.
Cell cycle analysis
Cell cycle analysis was performed using propidium iodide (PI). Briefly, cells were seeded in a 25 cm2 flask, then 60 % confluent cells were treated with MBIC for 24 and 48 h. Following incubation, the cells were harvested and spun down for 5 min at 2000 rpm. The supernatant was removed and cells were then fixed in 1 ml of ice-cold 70 % ethanol overnight at -80 °C. Fixed cells were washed with 1 ml 1X PBS and stained in 500 μl of PI containing 5 μg/ml DNase-free RNase for 30 min at room temperature in total darkness. DNA content of the cell was analyzed by flow cytometry. The percentage of G0-G1, S and G2-M cells were then calculated using Fluorescence-activated cell sorting (FACS) software (BD Biosciences).
Live-cell imaging
HeLa cells expressing EGFP-α-tubulin, EGFP-CENP-A and histone H2B-mCherry were grown in glass chambers (Thermo Scientific, USA). Thirty minutes before imaging, the medium was changed to pre-warmed Leibovitz’s L-15 medium (Life Technologies) supplemented with 20 % fetal bovine serum and 20 mM HEPES, pH 7.0. Recordings were made in a temperature-controlled incubator at 37 °C. Images were collected with an Olympus IX-71 inverted microscope (Olympus) controlled by DeltaVision softWoRx (Applied Precision) using a 20x 0.75 NA UPlan SApochromat objective lens (Olympus). Z-series of six sections in 3 μm increments were captured every 15 min. Image stacks were projected.
Tubulin polymerization assay
To investigate the effect of MBIC on tubulin polymerization, a fluorescence-based tubulin polymerization assay kit (Cytoskeleton-Cat. # BK011P) was used according to the manufacturer’s protocol. Briefly, 200 μl of pure tubulin protein was re-suspended in 420 μl of ice cold Tubulin Polymerization Buffer (TPB) to give a final concentration of 3 mg/ml tubulin, supplemented with 80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP, 20 % (v/v) glycerol. 100 μl of tubulin reaction mixture were added to each well in a 96-well plate containing the selected concentration of MBIC (10 μM). Paclitaxel, colchicine, nocodazole were applied separately as positive controls (10 μM). Samples were mixed well and the tubulin assembly was monitored by an increase in fluorescence emission at 340 nm in kinetic mode for 120 min at 37 °C using a plate reader (Infinite® 200 PRO—Tecan plate reader, USA).
Western blot analysis
Western blot analysis was used to investigate protein expression levels. It was adopted by Suresh Kumar et al. [20]. Briefly, 1 × 105 cells/flask was seeded and treated with MBIC in a dose-dependent manner for 24 h. Cells treated with colchicine and nocodazole served as positive controls. Cells were collected and lysed in RIPA buffer supplemented with a 10 μl protease inhibitor cocktail, sodium orthovanadate, and PMSF (Santa Cruz, USA). The lysate was stored at −80 °C until further use. A 40 μg of sample protein was resolved on 10 % SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, USA). The membrane was blocked in 5 % BSA for 1 h at room temperature following an overnight incubation at 4 °C with the following primary antibodies: Anti-Cyclin B1, anti-CDK1, anti-BubR1, anti-Aurora B, anti-cleaved PARP, anti-cleaved caspase-3/7/9, anti-Bcl-2 and anti-Bax (1:1000) (Cell Signaling Technology (CST), USA), mouse anti β-actin (1:40000) (Sigma-Aldrich, USA) antibodies.
Membranes were washed with 1X TBS-T prior to incubating with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies for 2 h at room temperature. Membranes were washed 3 times with 1X TBS-T to remove excess antibodies before the proteins-antibody complex was detected with Amersham ECL prime western blotting detection reagent (GE Healthcare, USA). Western blot images were quantified and processed by ImageJ software (NIH, USA).
Multiparameter cytotoxicity assay
The critical apoptotic events in cervical cancer including cell permeability, mitochondrial membrane potential (MMP), cytochrome c release and total nuclear intensity after treatment with MBIC were determined using the Cellomics Multiparameter Cytotoxicity 3 Kit. Briefly, 1 × 10 4 cells/well were seeded in a 96-well plate and incubated for 24 h prior to treatment with MBIC at various concentrations. At 24 h post treatment, cells were stained with cell permeability and MMP dye and further incubated for 1 h. Stained cells were fixed and permeabilized with 4 % formaldehyde and 0.1 % Triton X-100 in PBS, respectively. Cells were washed twice with 1X PBS prior to blocking with 3 % bovine serum albumin.
Cells were rinsed twice with Wash Buffer I (1X PBS) and incubated with 50 μl/well of Cytochrome c primary antibody. After 60 min, the plate was washed with 1X Wash Buffer and subsequently, 50 μl of goat anti-mouse secondary antibodies conjugated with DyLight 649 were added into each well. Cells were rinsed twice with Wash Buffer I (1X PBS). Hoechst 33342 was added into the staining solution to stain the nucleus. Stained cells in the 96-well plates were analyzed using ArrayScan High Content Screening (HCS) system (Thermo Scientific, USA).
Drug combination assay
In order to discover the synergistic effects of MBIC with conventional drugs briefly, cells were seeded in 1 × 104 cells/well and were incubated at 37 °C overnight. Thereafter, cells were treated with the selected conventional drugs—paclitaxel, colchicine, nocodazole and doxorubicin separately in various concentrations. Also, the MBIC, combined with each of the selected conventional drugs in a 1:1 ratio, were diluted in concentration from 50 μM to 0.1 μM. Cell viability assay (MTT assay) was performed 24 h post-treatment and absorbance were measured in a microplate reader to find out the IC50 for each conventional drug alone and in combination with MBIC on HeLa cells. For this analysis, we used the commercial software package CampuSyn (Biosoft, Cambridge, United Kingdom; Ref.9). After entering doses and effects of MBIC and other drugs either separately or combined, a normalized combination index (CI) and dose reduction index (DRI) with values of 50 % to 97 % inhibition was obtained. Furthermore, the software produced an automated isobologram and fraction affected-combination index (Fa-CI) plot.
Statistical analysis
Statistical analysis was processed according to conventional procedures using the Statistical Program for Social Sciences (SPSS) software for Windows, Version 12.0 (Post-hoc, Turkey’s test). A P value <0.05 was considered statistically significant.