Colorectal cancer cell line LoVo was provided by the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Colorectal cancer cells were incubated in DMEM/F-12 (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Life Technologies, Carlsbad, CA), 100 U/ml penicillin and 100 µg/ml streptomycin in 5% CO2 at 37 ℃. Cells in logarithmic growth phase were chosen for experiment.
Transwell assay for cell invasion.
For the cell-invasion test, 24-well transwells (pore size = 8 mm; Corning Incorporated, Corning, NY, USA) were coated with Matrigel (1 mg/mL) according to the manufacturer’s (Becton Dickinson) instructions. LoVo cells were suspended in the upper chamber (2 × 105 cells/well) of serum-free medium, and the lower chamber was 500 µL DMEM/F-12 medium containing 10% FBS. After 24 hrs of incubation with the indicated concentrations of paeonol (99% purity, Natura Pharmaceutical Co., Ltd. Zhejiang, China) or Celecoxib (Sigma-Aldrich, St. Louis, MO, USA), the cells of upper chamber were wiped clean by a cotton swab. Subsequently, the cells invading the lower chamber were fixed using 95% ethanol for 20 minutes, and then stained by 0.1% crystal purple for 10 minutes.
Wound-healing assay for cell migration.
Cell migration capacity was measured using a wound-healing assay. LoVo cells (1 × 106) were seeded in six-well plates and grew to form a confluent monolayer. a 200 µL micropipette tip was used to create a wound. The detached cells were then removed by washing with PBS and incubated with or without paeonol for 24 hrs. The photomicrographs were taken under the inverted microscope at 0, and 24 hrs of the same wound areas, and experiments were carried out in triplicate.
Determination of PGE 2 production.
The level of PGE2 in the cell culture supernatant was performed using the PGE2 ELISA kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. And the experiments were carried out in triplicate.
COX-2 siRNA synthesis and transfection.
LoVo cells (2 × 105 in 2 ml of DMEM/F-12 without antibiotics) were seeded in six-well plates. After 24 h, the COX-2 siRNA (Shanghai GenePharma Co., Ltd. Shanghai, China) mix with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was overlaid on the cells according to the manufacturer's instructions.
Western blot assay.
The cells were lysed with RIPA buffer supplemented with protease inhibitor. Total protein extract was separated on a 12.5% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel, and then transferred to nitrocellulose membranes using a wet transfer system. The membrane, after blocked by 5% fat-free milk, was incubated with primary antibodies: COX-2 (1:1000, Cell Signaling Technology), MMP-9 (1:1000, Cell Signaling Technology), E-cadherin(1:1000, Cell Signaling Technology), Vimentin (1:1000, Cell Signaling Technology), Fibronectin(1:1000, Cell Signaling Technology), Akt (1:1000, Cell Signaling Technology), p-Akt(1:1000, Cell Signaling Technology), ERK (1:1000, Cell Signaling Technology), p-ERK(1:2000, Cell Signaling Technology), and β-actin (1:1000, Cell Signaling Technology) at 4 ℃ for 12 hours. Following that, PVDF was then rinsed with TBST solution and incubated at room temperature for 1 hour with the secondary antibodies (Cell Signaling Technology) conjugated to HRP. The automatic developing instrument (ChemiDocXRS imaging system) was adopted to develop and the gray value was calculated.
All data were presented as Mean ± SD. GraphPad Prism 7.0 was adopted for statistical analysis of data. Student's t-test was used for comparison of the values between two groups. P < 0.05 indicated statistical significance.