Crizotinib powder was from Selleckchem Biotech, Inc. (Houston, TX, USA). 3–2,5-Diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Shanghai Solarbio Science & Technology Co., Ltd. (Shanghai, China). RPMI-1640 medium and PBS were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). RIPA lysate was purchased from Beyotime Institute of Biotechnology (Haimen, China). Cell apoptosis kit was from Solarbio. Transwell chamber was from Shanghai Yu Bo Biotechnology Co., Ltd. (Shanghai, China). Human non-small cell lung H2228 cell line was from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). Gel imaging system was from Thermo Fisher Scientific, Inc., (Waltham, MA, USA) and microplate reader was provided by Bio-Rad Laboratories, Inc., (Hercules, CA, USA). The study was approved by the Ethics Committee of Renji Hospital Shanghai Jiaotong University School of Medicine (Shanghai, China). Patients who participated in this research, signed the informed consent and had complete clinical data.

H2228 lung cancer cells containing the EML4-ALK fusion gene were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C under 5% CO₂. The medium was changed twice a week, and cells are harvested at logarithmic growth phase for subsequent experiments.

Cells were collected at logarithmic growth phase and inoculated into 96-well plates at a density of 1×10⁷/l, 200 µl for each well. Cells were cultured at 37°C and 5% CO₂ for 2 days in incubator. After cell adhesion, 0 (control group), 20, 40, 80, 160 and 320 nmol/l (treatment groups) crizotinib was added to treat the cells. After incubation for 3 days at 37°C and 5% CO₂, liquid was discarded and 8 µl MTT (5 g/l in PBS) was added to each well. After cell culture for another 4 h, cell culture was terminated. After centrifugation at 1,800 × g for 5 min at 4°C, culture supernatant was discarded, and 150 µl DMSO was added to each well. The absorbance (A) of each well was detected by microplate reader, and the cell inhibition rate and IC₅₀ value were calculated. Inhibition rate (%) = (1 - A value of treatment group/A value of control group) × 100%.

Cells were harvested at logarithmic growth phase, and cell density was adjusted to 5×10⁵/ml. Then 1 ml of cells was added into each well of 6-well plates. After cell adhesion, medium containing 300 nmol/l crizotinib was used to replace the original medium. After cell culture for 1, 2 and 3 days, cells were collected and stained, followed by flow cytometry (FACSCalibur; BD Biosciences, Detroit, MI, USA), to measure cell apoptosis rate. This experiment was performed three times.

Transwell chamber was placed in a 24-well culture plate, and a small amount serum-free medium was added into the upper chamber, followed by addition of Matrigel. The upper chamber was added with 400 µl of cell suspension (5×10⁴ cells/l), while the lower chamber was filled with 500 µl cell culture medium containing 10% fetal bovine serum. Cells were cultured under normal conditions, and 3 replicates were set for each experiment. After cell culture 24 h, Transwell chambers were collected and fixed with formaldehyde, followed by staining with 0.1% crystal violet. Ten visual fields were randomly selected under a microscope to calculate the average number of cells that penetrated the membrane.

RIPA solution was used to extract total protein from cells according to the instructions. Protein concentration was determined by BCA method. After blocking with 5% skimmed milk for 1 h, membranes were incubated with primary antibodies overnight at 4°C. After washing with TBST for 5 min ×3 times, membranes were incubated with secondary antibodies at room temperature for 1 h. Primary rabbit polyclonal JAK antibody (1:500; cat. no. ab47435), mouse monoclonal STAT antibody (1:500; cat. no. AMAB90777), rabbit polyclonal GAPDH antibody (1:500; cat. no. ab37168) and secondary goat anti-rabbit (HRP) IgG antibody (1:2,000; cat. no. ab6721) were all purchased from Abcam (Cambridge, MA, USA). Chemiluminescence method was used to detect the signal, and signal was scanned by a gel imager (Thermo Fisher Scientific, Inc.).

Statistical analysis was performed by using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Measurement data were expressed as mean ± SD. Comparison between multiple groups was done using One-way ANOVA test followed by post hoc test (Least Significant Difference). t-test was used for comparison between two groups, and repeated measurements variance analysis was used for intragroup comparison. P<0.05 was considered to indicate a statistically significant difference.

Cell proliferation was significantly inhibited after crizotinib treatment for 3 days, and the inhibitory rate increased with the increase of crizotinib concentration (p<0.01; Table I and Fig. 1). IC₅₀ value was 311.26 nnol/l after administration for 3 days. Crizotinib at a concentration of 300 nmol/l was used in follow-up experiments.

H2228 cells were cultured in medium containing 300 nmol/l crizotinib. After cell culture for 1, 2 and 3 days, cells were detected by flow cytometry. As shown in Fig. 2, crizotinib obviously promoted cell apoptosis (p<0.01). With the prolongation of time, apoptotic rate of cells treated with crizotinib was significantly increased. The apoptotic rate at the 1st day was obviously higher than that at 0 day (F=243.251, P<0.01); apoptotic rate at the 2nd day was obviously higher than that at the 1st day (F=279.783, P<0.01) and apoptotic rate at the 3rd day was obviously higher than that at 2nd day (F=167.524, P<0.01).

H2228 cells were cultured in medium containing 300 nmol/l crizotinib. Results of Transwell assay showed that the number of cells that infiltrated the membrane was significantly reduced with the prolonged treatment (p<0.01). The number of cells at the 1st day was obviously lower than that at 0 day (F=48.216, p=0.002); the number of cells at the 2nd day was obviously lower than that at the 1st day (F=68.398, p=0.001) and the number of cells at the 3rd day was obviously lower than that at the 2nd day (F=53.912, p=0.02) (Fig. 3).

Effect of crizotinib on the expression of JAK and STAT was detected by western blot analysis. Results showed that crizotinib significantly downregulated the expression of JAK protein after treatment for 1 day (p<0.01), but there was no significant difference at the 1st, 2nd, or 3rd day in the level of JAK protein (p=0.47) (Fig. 4). Crizotinib also significantly downregulated the expression of STAT protein after treatment for 1 day (p<0.01), but no significant difference was found either at the 1st, 2nd, or 3rd day in the level of STAT protein (p=0.91) (Fig. 5).