3.3. Biological Studies

Three cancer cell lines including human colon carcinoma (HCT116), human breast adenocarcinoma (MCF-7) and human hepatocellular carcinoma (Hep G2), were purchased from American Type Cell Culture Collection (ATCC, Manassas, VA, USA) and grown on the appropriate growth medium Dulbecco’s modified Eagle’s medium (DMEM) or Roswell Park Memorial Institute medium (RPMI 1640) supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin and 10% of heat-inactivated fetal bovine serum in a humidified, 5% (v/v) CO₂ atmosphere at 37 °C [34]. Cytotoxicity assay by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT).

Exponentially growing cells from different cancer cell lines were trypsinized, counted and seeded at the appropriate densities (2000–1000 cells/0.33 cm² well) into 96-well microtiter plates. Cells then were incubated in a humidified atmosphere at 37 °C for 24 h. Then, cells were exposed to different concentrations of compounds (0.1, 10, 100, 1000 µM) for 72 h. Then the viability of treated cells was determined using MTT technique as follow. Media were removed; cells were incubated with 200 μL of 5% MTT solution/well (Sigma Aldrich, Darmstadt, Germany) and were allowed to metabolize the dye into a colored-insoluble formazan crystal for 2 h. The remaining MTT solution were discarded from the wells and the formazan crystals were dissolved in 200 µl/well acidified isopropanol for 30 min, covered with aluminum foil and with continuous shaking using a MaxQ 2000 plate shaker (Thermo Fisher Scientific Inc, Waltham, MA, USA) at room temperature. Absorbance were measured at 570 nm using a Stat Fax^R 4200 plate reader (Awareness Technology, Inc., Awareness Technology, FL, USA). The cell viability was expressed as percentage of control and the concentration that induces 50% of maximum inhibition of cell proliferation (IC₅₀) were determined using Graph Pad Prism version 5 software (Graph Pad Software Inc, San Diego, CA, USA) (1,2) [35].

The in-vitro enzyme inhibition determination for the synthesized compounds was carried out at Bio Science Corporation (BPS)-San Diego, CA, USA. The assay was performed using Kinase-Glo Plus luminescence kinase assay kit (Promega), where the kinase activity was measured by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity. Our compounds were diluted with 10% DMSO and 5 µL of the dilution was added to a 50 µL reaction so that the final concentration of DMSO is 1% in all of reactions. All of the enzymatic reactions were conducted at 30 °C for 40 min. The 50 µL reaction mixture contains 40 mM Tris, pH 7.4, 10 mM MgCl₂, 0.1 mg/mL BSA, 1 mM DTT, 10 µM ATP, Kinase substrate and the enzyme. After the enzymatic reaction, 50 µL of Kinase-Glo Plus Luminescence kinase assay solution (Promega San Diego, CA, USA) was added to each reaction and incubate the plate for 5 min at room temperature. Luminescence signal was measured using a BioTek Synergy 2 microplate reader.

Kinase activity assays were performed in duplicate at each concentration. The luminescence data were analyzed using the computer software, Graphpad Prism. The difference between luminescence intensities in the absence of Kinase (Lu_t) and in the presence of Kinase (Lu_c) was defined as 100% activity (Lu_t − Lu_c). Using luminescence signal (Lu) in the presence of the compound, % activity was calculated as:

where Lu = the luminescence intensity in the presence of the compound.

HCT116 cells at a density of 4 × 10⁶ cell/T 75 flask were exposed to 1-(4-Chlorophenyl)-3-(4-((3-methylquinoxalin-2-yl)amino)phenyl)urea VIIIc at its IC₅₀ concentration for 24 and 48 h. The cells then were collected by trypsinization, washed with phosphate buffered saline (PBS), and fixed in ice-cold absolute alcohol. Thereafter, cells were stained, using Cycle test ^TM Plus DNA Reagent Kit (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instructions. Cell cycle distribution was determined using a FACS Calibur flow cytometer (BD Biosciences).

Apoptosis was determined by staining cells with Annexin V–fluorescein isothiocyanate (FITC) and counterstaining with propidium iodide (PI) using the Annexin V–FITC/PI apoptosis detection kit (BD PharMingen, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, 4 × 10⁶ cell/T 75 flask were exposed to compound VIIIc at its IC₅₀ concentration for 24 and 48 h. The cells then were collected by trypsinization and 0.5 × 10⁶ cells were washed twice with phosphate-buffered saline (PBS) and stained with 5 μL Annexin V–FITC and 5 μL PI in 1× binding buffer (BD PharMingen) for 15 min at room temperature in the dark. Analyses were performed using FACS Calibur flow cytometer (BD Biosciences).

Cell Line cells were obtained from American Type Culture Collection, cells were cultured using DMEM (Invitrogen/Life Technologies, Waltham, MA, USA) supplemented with 10% FBS (Hyclone, Waltham, MA, USA), 10 μg/mL of insulin (Sigma), and 1% penicillin-streptomycin. All of the other chemicals and reagents were from Sigma, or Invitrogen. Plate cells (cells density 1.2–1.8 × 10,000 cells/well) in a volume of 100 µL complete growth medium + 100 μL of the tested compound per well in a 96-well plate for 24 h before the MTT assay.