br Soft agar colony formation assay
2.5.3. Soft agar colony formation assay
A 6-well plate was coated with a 1:1 ratio of 1.2% agarose and 2× DMEM that was allowed to solidify for 30 min. The top portion was pre-pared with 0.6% agarose and 2× medium, and SKOV3 and HO-8910 cells that had been treated with or without apatinib (20 μM) were plated at a density of 2000 cells/mL. Images were captured after 14 days of culture.
2.6. Western blotting
SKOV3 and HO-8910 cells were seeded onto six-well plates at a den-sity of 3 × 105 cells per well. Following a 24-h incubation, cells were treated with 20 μM apatinib for 24 h. Then, the cells were harvested, washed twice with pre-chilled PBS, lysed with cell lysis buffer (Beyotime Biotechnology, CHINA) containing 10 mM phenylmethanesulfonyl fluoride (PMSF; Sigma, USA), an EDTA-free protease inhibitor cocktail (Roche, Germany) and PhosSTOP™ (Roche, Germany) for 30 min on ice, and centrifuged at 12,000 rpm for 15 min at 4 °C. The protein concentrations of the supernatants were quantified using a BCA protein assay kit (TIANGEN BIOTECH, CHINA), and equal amounts of protein from each sample were loaded onto gels and sepa-rated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After separation was complete, the proteins in the gel were transferred onto polyvinylidene fluoride (PVDF) membranes (0.45 μm; Millipore, USA) and blocked with 5% Difco Skim Milk (BD, USA) for 2 h at room temperature. Membranes were subsequently incu-bated with the indicated primary Pyocyanin at 4 °C overnight. On the next day, membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST, pH 7.4) and incubated with the appropriate sec-ondary antibody at room temperature for 1 h. The immunoblots were detected by enhanced chemiluminescence (ECL, Beyotime Biotechnol-ogy, CHINA), and the band densities were analysed using an ImageQuant LAS 4000 system (GE, USA). r> 2.7. Mouse xenograft model
All experiments conformed to the “Guidelines and suggestions for the care and use of laboratory animals” (Ministry of Science and Tech-nology of the People's Republic of China, 2006). The rats were main-tained under standard laboratory conditions (20–22 °C, 50–70% relative humidity, 12:12-h light:dark cycle) and had free access to food and water before and after the surgical procedures.
BALB/c-nu/nu mice aged 4–6 weeks and weighing 16–20 g were used to establish the SKOV3 xenograft model. The mice were purchased from Sino-British SIPPR/BK Lab Animal Co., Ltd. (Shanghai, China) and maintained in pathogen-free cages with standard rodent chow and ster-ile water available ad libitum. Briefly, 5 × 106 SKOV3 cells were suspended in 200 μL of PBS and inoculated subcutaneously into the right flanks of the nude mice. When the tumours reached a mean diam-eter of 6 mm, the mice were divided randomly into two groups: control (10% DMSO) and apatinib (50 mg/kg). The mice were intragastrically administered control or apatinib solutions every other day. Throughout the treatment period, the mice were weighed, and their tumours were measured with callipers every 3 days. Tumour volume (V) was calcu-lated using the following formula: V = largest diameter × (smallest diameter)2/2 mm3. After 40 days of treatment when the tumours had grown to a proper size, the mice were sacrificed, and the tumours
Days after Tumour Inoculation
Fig. 5. A. Photograph of tumours from the vehicle-treated (upper tissues) and apatinib-treated groups (lower tissues). B. Tumour sizes of SKOV3 ovarian cancer xenografts from mice treated with vehicle (blue curve) and 50 mg/kg apatinib (orange curve).
were excised, weighed and stored at −80 °C or placed in 4% paraformal-dehyde for further examination.
2.8. Statistical analysis
Each experiment was performed at least three times. Statistically significant differences among groups were evaluated with t-tests using SPSS18.0 software. P b 0.05 and P b 0.01 were considered to rep-resent different levels of statistical significance.
3.1. Apatinib does not appreciably affect the proliferation and vitality of ovarian cancer cells
3.2. Apatinib inhibits the migration of ovarian cancer cells
We performed wound-healing, transwell and soft agar assays to de-termine the anti-tumour activity of apatinib on ovarian cancer cell mi-gration. Apatinib decreased the migratory capacities of SKOV3 and HO8910 ovarian cancer cells at concentrations of 10 μM and 20 μM (Fig. 2A) and 20 μM (Fig. 2B). We then used a soft agar growth assay to investigate the effect of apatinib on the tumourigenesis of SKOV3 and HO8910 ovarian cancer cells. Fewer SKOV3 and HO8910 cell colo-nies were observed after the 20-μM apatinib treatment than after the control treatment (Fig. 2C). As shown in Fig. 2A–C, the migration and in-vasion of both SKOV3 and HO8910 cells were significantly reduced fol-lowing treatment with apatinib (P b 0.01).