941678-49-5 br The results are expected to provide experimen
The results are expected to provide experimental data and a theoretical basis for the use of the anti-ovarian cancer agent apatinib in clinical practice.
2. Materials and methods
Apatinib was obtained from Hengrui Medicine Co., Ltd. (Jiangsu, China). For the in vitro studies, apatinib was dissolved in 100% dimethyl sulfoxide and diluted to the desired concentration with Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, USA). For the in vivo studies, apatinib was diluted in phosphate-buffered saline.
The human ovarian cancer cell lines SKOV3 and HO-8910 were ob-tained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (Gibco, USA) containing 10% foetal bovine serum (FBS; Gibco, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (penicillin-streptomy-cin-glutamine; Gibco, USA) at 37 °C in a humidified 941678-49-5 of 5% CO2 in a Forma Steri-Cycle CO2 Incubator (Thermo Fisher Scientific, Massachusetts, USA).
2.3. Cell proliferation assay
medium was replaced with 100 μL of medium containing 10 μL of MTT (5 mg/mL), and the cells were incubated for an additional 4 h. The ab-sorbance of the solution was measured at 490 nm using a microplate reader (Varioskan Flash, THERMO, USA).
The effects of apatinib on the cell cycle and apoptosis were detected by flow cytometry.
A flow cytometry analysis was conducted to assay cell cycle arrest in SKOV3 and HO-8910 cells treated with 20 μmol/L apatinib. SKOV3 and HO-8910 cells were seeded onto six-well plates at a density of 3 × 105 cells per well. Following a 24-h incubation, cells were treated with 20 μM apatinib for 24 h and then subjected to cell cycle assays. Cells were harvested and washed twice with pre-chilled PBS, followed by fixation with 70% ethanol at 4 °C for 2 h. After washes with PBS, cells were stained with 1 μg/mL propidium iodide (PI) (THERMO, USA) in 1 mL of PBS at 4 °C for 30 min. The stained cells were analysed using a flow cytometer (FACS Aria II, BD Biosciences, USA).
SKOV3 and HO-8910 cells were seeded onto six-well plates at a den-sity of 3 × 105 cells per well. After a 24-h incubation, cells were treated with 20 μM apatinib for 24 h and then subjected to apoptosis assays. Cells were harvested and washed twice with pre-chilled PBS. Then, a Dead Cell Apoptosis Kit with Annexin V Alexa Fluor® 488 and PI was used to stain the cells for 15 min at room temperature according to the manufacturer's instructions. The stained cells were analysed using a flow cytometer (FACS Aria II, BD Biosciences, USA). FlowJo software was used to analyse and process the results.
Fig. 2. A Apatinib significantly suppressed SKOV3 and HO8910 cell migration, as assessed using wound-healing assays (**P b 0.01). B Apatinib significantly suppressed SKOV3 and HO8910 cell migration, as determined using transwell assays (**P b 0.01). C Apatinib significantly suppressed SKOV3 and HO8910 cell migration, as determined using soft agar assays (**P b 0.01).
Changes in ovarian cancer migration in vitro after apatinib treatment were detected using scratch wound-healing, transwell and soft agar assays.
SKOV3 and HO-8910 cells were seeded in six-well plates at a density of 5 × 105 cells per well and allowed to reach 100% confluence prior to the wound-healing assays. The cell layer was wounded with a pipette
Fig. 4. After a 24-h treatment with 20 μM apatinib, the levels of proteins involved in the JAK/STAT3, PI3K/Akt, and Notch signalling pathways, including JAK, p-JAK, Stat3, p-Stat3, PI3K, Akt, p-Akt, Notch1, Hes1 and Hey1, in Skov3 and HO-8910 cells were determined by western blotting.
tip, and the floating cells were removed by two washes with PBS. Cells were then cultured in serum-free DMEM in the presence or absence of apatinib (10 μM and 20 μM) for 24 h, during which the cells were im-aged using a live cell imaging system (Zeiss, German). Wound healing was assessed by measuring the distance between the lines of the wounded area under a microscope at 100× magnification.
2.5.2. Transwell migration assay
Twenty-four-well transwell chambers (8-μm pore size, 6.5-mm di-ameter) (Millipore, USA) were used to assay migration and invasion. SKOV3 and HO-8910 cells that had been treated with or without
apatinib (20 μM) were harvested and resuspended in serum-free DMEM at a density of 2 × 105 cells/mL. Next, 100-μL cell suspensions were seeded into the upper chambers of the transwell plates, and 500 μL of DMEM containing 10% FBS were placed in the lower chambers. After co-culture for 24 h, cells on the upper surfaces of the transwell membranes were removed using cotton swabs, and those on the lower surfaces were fixed with ice-cold methanol and stained with a 0.1% crystal violet solution. The number of migrated cells was counted in five random fields.