br Two NSCLC cell lines A and NCI H and
Two NSCLC cell lines A549 and NCI-H1650, and two human normal cell lines BEAS-2B and L02 (human bronchial epithelial cell line BEAS-2B; human liver cell line L-02) were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). A549 and BEAS-2B CCK8 were maintained in DMEM (HyClone) medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin. NCI-H1650 and L-02 cells were cultured in RPMI-1640 (HyClone) medium supplemented with 10% FBS, 1% penicillin and streptomycin. All the cells were cultured at 37 °C in a 5% CO2 humidified atmosphere.
2.3. Cell viability assay
Cell viability upon treatment of the compounds was determined by using Cell Counting Kit-8 assay (Sangon Biotech, Shanghai, China) following the manufacturer's instructions. Cells were seeded in 96-well plates at density of 5 × 103 per well and were incubated for 72 h with either vehicle (DMSO) or desired concentrations of compounds at 37 °C in a 5% CO2 incubator. Then, cells were incubated with CCK-8 solution for 1 h at 37 °C in a 5% CO2 incubator. Finally, the absorbance at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Shanghai, USA). Cell viability was calculated as the ratio of the ab-sorbance of the treated cells to the absorbance of the control groups. IC50 values were calculated by GraphPad Prism 6, which were the mean values derived from three independent experiments. Life Sciences 219 (2019) 20–30
2.4. Colony formation assay
Colony formation assay was used to evaluate the anti-proliferative eﬀect of CAA45 against A549 and NCI-H1650 cells as previously re-ported . Varying cell numbers were seeded in 6-well plates and incubated at 37 °C for 24 h. Then the cells were treated with either DMSO or diﬀerent concentrations of CAA45 (0.01, 0.03 and 0.06 μM) for 24 h, and then replaced by compound-free media for every 3 d. After 14 d cell plating, cell colonies were fixed with 4% paraformaldehyde for 15 min, and stained by 0.1% crystal violet for 30 min at room tem-perature. Colonies with over 100 cells were defined as positive.
2.5. DNA Top I inhibition assay
The inhibition of CAA45 on DNA Top I was performed indirectly by evaluating the relaxation of supercoiled pBR322 plasmid DNA. The experimental conditions were as previously reported . Specifically, each reaction mixtures contained 2 μL 10× DNA Topo I buﬀer, 2 μL 0.1% BSA, 0.5 U Topo I protein, 0.25 μg of pBR322 plasmid DNA (Ta-kara, Japan) and the test compound CAA45 (1, 5, and 10 μM) or posi-tive control CPT (while the drug was omitted in control panel (panel 2) representing the capability of Topo I activity), then deionized water was used to complement the reaction volume to 20 μL. The reaction was performed at 37 °C in a water bath for 30 min and stopped by adding 1 μL 10% SDS and 3.5 μL 6× loading buﬀer. Finally, the reaction mixtures were separated by electrophoresis on a 1% agarose gel at 100 V for 40 min. The gels were stained with 0.5 mg/mL ethidium bromide for 15 min and visualized by using Molecular Imager FX (Biorad, Hemel Hempsted, UK).
2.6. Wound healing assay
A549 cells were seeded in 6-well plates at density of 1 × 106 cells/ well. After reaching nearly confluence, a wound was created by using a p200 pipette tip in the middle area of confluent cells and then FBS free medium was used to remove the detached cells. Then cells were treated with either vehicle or CAA45 at the desired concentrations at 37 °C for 24 h. Images were taken by using phase-contrast microscopy (Olympus, IX51, Tokyo, Japan). The cell migration area was then calculated by ImageJ software (the formerly scratch area minus the final vacant area between cells). The ratio of migration area to the formerly scratched area indicates how many A549 cells migrate.
2.7. Transwell migration assay
Cells were suspended in serum-free DMEM and transferred on the top of transwell chambers (1 × 105 cells per Transwell) (Corning, New York, USA). DMEM with 10% fetal calf serum was placed in the lower chambers in the presence or absence of CAA45. After incubation for 24 h, the migrated cells in the bottom of the chamber were fixed by using 90% alcohol and stained with crystal violet staining solution (Beyotime, Jiangsu, China). Images were taken by using phase-contrast microscopy (Olympus, IX51, Tokyo, Japan). Five random fields per well were counted.