3.3. Biological Test Method

The wild-type EGFR kinase enzyme system (Catalog. V3831) and the T790M/L858R-mutated EGFR kinase enzyme (Catalog. V5324) were purchased from Promega Corporation (Fitchburg, WI, USA). Concentrations consisting of suitable levels from 0.1 to 100 nM were used for all of the tested compunds. The experiments were performed according to the instructions of the manufacturer. The more detailed and complete protocols, see the ADP-Glo™ kinase Assay Technical Manual #313, and the active kinase datasheet available at: http://www.promega.com/tbs/tm313/tm313/tm313.html and http://www.promega.com/KESProtocol (or http://www.promega.com/tbs/signaling.htm), respectively. The test was performed in a 384-well plate, and includes the major steps below: (1) perform a 5 μL kinase reaction using 1× kinase buffer (e.g., 1× reaction buffer A); (2) incubate at room temperature for 60 min; (3) add 5 μL of ADP-Glo™ Reagent to stop the kinase reaction and deplete the unconsumed ATP, leaving only ADP and a very low background of ATP; (4) incubate at room temperature for 40 min; (5) add 10 μL of Kinase Detection; (6) reagent to convert ADP to ATP and introduce luciferase and luciferin to detect ATP; (7) incubate at room temperature for 30 min; (8) plate was measured on TriStar^® LB942 Multimode Microplate Reader (BERTHOLD TECHNOLOGIES GmbH & Co. KG., Bad Wildbad, Germany) to detect the luminescence (Integration time 0.5–1 s). Curve fitting and data presentations were performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.).

All the cell viability assays were performed according to the CCK-8 method. The cells were seeded at a density of 5 to 8 × 10⁴ cells/mL in 96-well plates in growth medium supplemented with 10% serum at 37 °C with 5% CO₂ for one day. After 12 h of incubation, 100 μL of medium was removed, and 100 μL of sample solution with different concentrations of inhibitor was added and then the cells were incubated for 48 or 72 h. Subsequently, 10 μL of CCK-8 reagent (Biotool Company, Kirchberg, Switzerland, 5.0 mg/mL) dissolved in phosphate-buffered saline (PBS) was added and the cells were incubated for another 4 h. The absorbance was read at 450 nm with a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The data were calculated using GraphPad Prim version 5.0.

Approximately 2 × 10⁵ cells/well of A431 and H1975 cells in 6-well plates were incubated in an incubator for 48 h, then treated with different concentrations of inhibitors for 48 h. After incubation, the cells were washed with PBS twice. Then, total of 20 μL of the solution containing the AO/EB dye mix (1.0 μg/mL of AO and 1.0 μg/mL of EB in PBS) was added to the cells. The apoptotic, necrotic, and live cells were observed and counted under the fluorescent microscope (OLYMPUS, Tokyo, Japan). DAPI staining was performed after being treated as mentioned above. The cells plated in 6-well plates were washed twice with PBS and fixed with 10% formaldehyde for 10 min, then washed with PBS three times. Cells were subsequently incubated in DAPI (1.0 μg/mL) solution at room temperature for 10 min, washed with PBS and examined under a fluorescence microscope (OLYMPUS).

The cancer cells were cultured in 6-well plates for 48 h at 37 °C. Wounds were created in the cell monolayer and washed with PBS to remove cell debris, then the cells were treated with different concentrations of inhibitor for 48 h. After that, the dead cells were washed away with PBS, and the images were taken by the fluorescence microscope (OLYMPUS).

Cell migration assay was evaluated by using Boyden chambers containing a transwell membrane filter with an 8 μm size pore (Corning Costa Corp, Cambridge, MA, USA). Prior to the invasion assay, the filter membrane was coated with 60 μL of Matrigel (BD Biosciences, Billerica, MA, USA) at a 1:8 dilution and rehydrated by adding 0.5 M serum-free medium to the apical side of the chamber at 37 °C for 0.5 h. The cells (2 × 10⁵ cells/well for invasion assay) were seeded to the apical side of the chamber with 200 μL medium with different concentrations of inhibitors, and the basolateral side of the chamber was filled with 600 μL medium containing 10% FBS. After 24 h at 37 °C, the cells adherent to the upper surface of the filter were swept by cotton swabs, then fixed with methanol, stained with crystal violet, and the cells were counted under a microscope in five random fields, irrespective of staining intensity. The images were taken by the fluorescence microscope (OLYMPUS).

The human H1975 and A431 cells at a density of approximately 2 × 10⁵ cells/well were plated in a 6-well plate and then treated with different concentrations of inhibitors for 24 h. Then the cells were harvested, resuspended in 1 mL of DCFH-DA (10 mM), and the levels of ROS were determined by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA).

The H1975 and A431 cells (1 to 5 × 10⁵ cells/well) incubated in 6-well plates were treated by different concentrations of inhibitors for 48 h. Then, they were collected and fixed with 70% ethanol at 4 °C overnight. After beening fixed with 75% ethanol at 4 °C for 24 h, the cells were stained with Annexin V-FITC (5 μL)/propidium iodide (5 μL) and analyzed by flow cytometry assay (Becton-Dickinson, Franklin Lakes, NJ, USA).

The H1975 and A431 cells at a density of approximately 2 × 10⁵ cells/well were incubated in 6-well plates, treated with different concentrations of inhibitors for 48 h, collected and fixed with 70% ethanol at 4 °C overnight. After fixation, the cells were washed with PBS and stained with propidium iodide (PI) for 10 min under subdued light. Stained cells were analyzed by flow cytometry assay (Becton-Dickinson, Franklin Lakes, NJ, USA).

The Roche Accu-chek^® Go blood meter was used for the determination of glucose concentration. Before testing, standard set-up of blood meter was performed according to the instrument’s specifications. The meter displays the blood sugar level in units of mmol/L. Male SD rates (180–220 g) were fasted overnight prior to dosing the next morning. Water was allowed ad libitum throughout the study. Rats were orally given compound DY3002 (50 mg/kg) and a vehicle. For the oral study, the compounds were formulated as a solution (Solutol HS15: NS = 30:70, v:v). Sampling occurred prior to dosing and at 7–9 different time points up to 12 h. Concentration of glucose in the blood was determined by blood meter.