br according to the manufacturer s protocol The
according to the manufacturer’s protocol. The culture medium was centrifuged at 3000×g for 15 min to remove cells and debris. Then, the supernatants were transferred to new tubes, and the reagents were added. After overnight incubation, the samples were centrifuged at 10,000 × g for 30 min. The exosomes were pelleted and resuspended in
Fig. 2. Aspirin inhibits the hypoxia-enhanced stemness of A549 cells. (A–C) mRNA levels of SOX-2, OCT4, and ALDH1 were determined by qRT–PCR using specific primers, with GAPDH serving as the internal control. (D, E) mRNA levels of HIF-1α and COX-2 were determined by qRT–PCR using specific primers, with GAPDH serving as the internal control. (F) The concentration of PGE2 secreted by A549 cells was determined by ELISA. Each bar indicates the mean ± SD of n = 3 experiments. * indicates P < 0.05, ** indicates P < 0.01. (G) SOX-2, OCT4, ALDH1, HIF-1α, and COX-2 protein levels were determined by Western blot, with GAPDH serving as the loading control.
PBS. The total protein concentration in exosomes was assessed using BCA Protein Assay Kit (KeyGEN Biotech). The number of exosomes from each sample was quantified by the EXOCET exosomes quantifi-cation assay kit (System Biosciences). The colorimetric assay at 405 nm was performed along with the standard curve for quantification.
2.9. Transmission electron microscopy (TEM)
Exosomes were isolated and resuspended in 100 μl PBS, and 50 μl of the suspension was placed onto formvar carbon-coated copper grids at room temperature for 20 min. The excess suspension was removed using filter paper. Exosomes were stained with 2% phosphotungstic 3-Cysteinylacetaminophen (PTA) at room temperature for 1 min. The grids were fixed with 2% glutaraldehyde at room temperature for 5 min. Images were obtained with a transmission electron microscope (JEM-1230, Jeol Ltd, Japan).
2.10. Transwell migration assay
A549 cells were treated with PBS or different exosomes for 24 h. Cells were collected after 24 h incubation with serum-free medium. 5 × 104 cells were resuspended in serum-free medium and placed in inserts. These inserts were then placed in wells with the serum-con-taining medium. Cells were cultured for an additional 12 h, and the top cells were removed using a cotton wool swab. The cells on the bottom surface were then fixed with 4% formaldehyde and stained with crystal violet for 30 min. The cells were captured and counted under an in-verted microscope (Leica, Germany).
2.11. In vitro angiogenesis experiment
Matrigel (BD Biosciences) was thawed at 4℃ overnight, was diluted and layered in a 24-well plate, followed by incubation at 37℃ for 2 h to allow polymerization. HUVEC cells (2 × 104 cells/well) were plated onto the Matrigel layer and incubated with exosomes for 24 h. The tube-like structure was visualized and imaged under an inverted mi-croscope (Leica). The number of branches and tube length were quan-tified by ImageJ software.
After aspirin or GW4689 (Sigma-Aldrich, USA) treatment, A549 cells were treated with vehicle or 2 μg/ml cisplatin (DDP) for 24 h. Annexin-V-FITC/PI staining was performed using Annexin-V-FITC/PI Apoptosis Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. Briefly, cells were har-vested and washed twice with PBS and then resuspended in the binding buffer. The 100 μl cell solution (1 × 105 cells) was incubated with 5 μl Annexin-V-FITC for 15 min in darkness, followed by incubation with 5 μl PI for 15 min. The samples were analyzed by flow cytometry (BD Biosciences).
2.13. Statistical analysis
All experiments were repeated at least three times, and data were presented as mean ± SEM. The data were analyzed by one-way ana-lysis of variance (ANOVA). Statistical comparisons between two groups were estimated using Student’s t-test. P < 0.05 was considered statis-tically significant. All statistical analyses were calculated by using SPSS 19.0 statistics software (IBM).
3.1. Aspirin inhibits tumorigenesis in A549 cells under hypoxic conditions
To evaluate the effects of aspirin on the tumorigenic properties of cells under hypoxic conditions, cell proliferation was monitored by CCK-8 assay. As shown in Fig. 1A, hypoxia-induced transient cell growth arrest within 24 h of treatment followed by rapid cell pro-liferation in A549 cells. Aspirin significantly inhibited cell growth in a dose-dependent manner compared with that observed in the corre-sponding control cells (Fig. 1A). Further, we analyzed the cell cycle by flow cytometry (FACS). After 24 h of culture, aspirin remarkably in-creased the percentage of G2/M phase cells (P < 0.05) in comparison with the hypoxia group (Fig. 1B, C). These findings indicate that aspirin inhibits cell growth and induces cell cycle arrest at the G2/M phase in hypoxic A549 cells.