Aspirin inhibits tumor progression and enhances cisplatin sensitivity in epithelial ovarian cancer

Cell culture

The EOC cell lines A2780, Caov-3 and SK-OV-3 were purchased from the China Center for Type Culture Collection (CCTCC). A2780 and Caov-3 are ovarian adenocarcinoma cell lines derived from malignant tumor tissues of untreated patients with ovarian cancer. SK-OV-3 is an ovarian adenocarcinoma cell line derived from malignant ascites. All cell lines were maintained at 37 °C in DMEM/F12 (Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) in a 5% CO₂ atmosphere. Aspirin (Sigma, St. Louis, MO, USA) was added for the indicated times after 12-24 h of cell growth. All assays below were performed in triplicate.

Cell viability assays

Cells were seeded at a density of 5000 cells/well in 96-well tissue culture plates. Different concentrations of aspirin (0.2 mM, 0.4 mM, 0.8 mM, 1.6 mM, 3.2 mM, or 6.4 mM) were added to the culture media after cell attachment, and five replicate wells for each concentration were established. Forty-eight hours after treatment, 20 µl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were added to the media, and the cells were further incubated for 4 h. Subsequently, the cells were solubilized in 100 µl of solubilization solution for 8 h. The optical density (OD) at 570 nm was measured using a microplate reader (Bio-Rad, Berkeley, CA, USA).

Cell proliferation assays

Cells were seeded in a 96-well tissue culture plate (Corning Costar, Corning, NY, USA) and adjusted to a density of 5000 cells/well. After treatment with different concentrations of aspirin (0 µM, 100 µM, and 1,000 µM) and/or CDDP (20 µM), cells were fixed and stained with an EdU cell proliferation kit (RiboBio Co., Guangzhou, China). Cells were immediately observed under a fluorescence microscope (Olympus, Tokyo, Japan), and cells in three random fields per well were counted (100 ×).

Colony formation assays

Cells were seeded in 6-well tissue culture plates in triplicate, and the density was adjusted to 500 cells/well. Cells were cultured in DMEM/F12 supplemented with 10% FBS and different concentrations of aspirin (0 µM, 25 µM, 100 µM, 500 µM, 1,000 µM, and 25 µM) and/or CDDP (5 µM) at 37 °C in a 5% CO₂ incubator (Heraeus, Hanau, Germany) for 14 days. Next, cells in the wells were fixed with methanol and stained with 0.1% crystal violet; the cell colonies formed were then visible to the naked eye.

Flow cytometry analysis

Cells were plated in triplicate in a six-well plate at a density of 1 ×10⁵ cells/well and incubated for 24 h. Then, cells were treated with different concentrations of aspirin (0 µM, 100 µM, and 1,000 µM) and/or CDDP (20 µM). After a 48-h incubation at 37 °C, cells were harvested, trypsinized and washed with cold phosphate-buffered saline (PBS). Subsequently, cells were stained with APC Annexin V and propidium iodide (PI) (BioLegend, Inc., San Diego, CA, USA). The stained cells were immediately analyzed using an LSR flow cytometer (Beckman Coulter, Inc., 250 S. Kraemer Boulevard, Brea, CA, USA), and the data were analyzed using Summit 6.2 software (Beckman Coulter, Inc., 250 S. Kraemer Boulevard, Brea, CA, USA).

Western blot analysis

After treatment with different concentrations of aspirin (0 µM, 100 µM, and 1,000 µM) for 24 h, cells were collected in cold Nonidet P40 (NP40) buffer containing a protease inhibitor cocktail for 30 min and centrifuged at 13000 × g for 10 min; subsequently, the supernatants were collected. The protein concentration was measured with a bicinchoninic acid (BCA) assay. Proteins in the samples were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing Tween 20 (TBST) for 1 h at room temperature and incubated with rabbit anti-p53 (1:1500; Cell Signaling Technology, Boston, MA, USA), rabbit anti-acetylated p53 (K382) (1:500; Abcam, Cambridge, UK), and mouse anti- β-actin (1:4000; Cell Signaling Technology, Boston, MA, USA) antibodies at 4 °C overnight. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:5000; Cell Signaling Technology, Boston, MA, USA) were used for detection as appropriate. Immunoreactions were visualized with an enhanced chemiluminescence kit (Bio-Rad, Berkeley, CA, USA), and the gray values of the bands were quantified with Quantity One Software (Bio-Rad, Berkeley, CA, USA) or a Molecular Imager ChemiDoc™ XRS ₊ system with Image Lab™ software (Bio-Rad, Berkeley, CA, USA).

Quantitative real-time polymerase chain reaction (qPCR)

After treatment with different concentrations of aspirin (0 µM, 100 µM, and 1,000 µM) for 24 h, total cellular RNA was extracted from cultured cells with TRIzol reagent (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s protocol. The quality and quantity of total RNA were evaluated with a NanoDrop 2000/2000C spectrophotometer (Thermo Fisher, Waltham, MA, USA), and cDNAs were subsequently synthesized using a reverse transcription kit (Takara, Tokyo, Japan) and the following primers: CDKN1A, upstream 5′-CGATGGAACTTCGACTTTGTCA-3′ and downstream 5′-GCACAAGGGTACAAGACAGTG-3′; BAX, upstream 5′-CCCGAGAGGTCTTTTTCCGAG-3′ and downstream 5′-CCCGAGAGGTCTTTTTCCGAG-3′; FOXF1, upstream 5′-GCGGCTTCCGAAGGAAAT-3′ and downstream 5′-CAAGTGGCCGTTCATCATGC-3′; PUMA, upstream 5′-CAAGTGGCCGTTCATCATGC-3′ and downstream 5′-CAAGTGGCCGTTCATCATGC-3′; RRAD, upstream 5′-CAAGTGGCCGTTCATCATGC-3′ and downstream 5′-CAAGTGGCCGTTCATCATGC-3′; and β-actin, upstream 5′-GCCAACACAGTGCTGTCTGG-3′ and downstream 5′-GCTCAGGAGGAGCAATGATCTTG-3′. β-Actin expression was used for normalization. PCR was performed in an Applied Biosystems StepOnePlus Real-time PCR system (Thermo Fisher, Waltham, MA, USA) using SYBR Green Real-time PCR Master Mix (Takara, Tokyo, Japan). The reactions were performed with a two-step thermal cycling method, 95 °C for 30 s and 40 PCR cycles of 95 °C for 5 s and 66 ° C for 30 s, followed by a melting curve analysis. The relative expression levels were calculated using the Ct (2 ^−ΔΔCt) method.

Orthotopic xenograft mouse model of human EOC

The Institutional Animal Care and Use Committee of Tongji Medical College approved this research ([2014] IACUC Number: 399). Six-week-old female BALB/c-nu/nu mice were purchased from Beijing HFK Bioscience Co. and were housed in sterile cages with ad libitum access to food in a specific pathogen-free (SPF) barrier animal room at Tongji Medical College Experimental Animal Center. A2780-Luciferase cells were obtained by transfecting the EOC cell line A2780 with a lentivirus carrying the luciferase gene. A2780-Luciferase cells were suspended in serum-free medium, and the density was adjusted to 1 ×10⁷ cells/ml. A 200-µl aliquot of the cell suspension was subcutaneously injected into the right axilla of nude mice. When the tumor volume reached approximately one cm³, mice were anesthetized with isoflurane, and the tumor was harvested. Subsequently, the tumor was cut into fragments with a volume of approximately 1-2 mm³. Eighteen mice were randomly divided into three groups according to a random number table (n = 6). Then, six mice per group were inoculated with one tumor fragment in the left ovary. Mice in the CDDP group were treated with CDDP by intraperitoneal injection and with water by gavage, mice in the CDDP plus aspirin group were treated with CDDP by intraperitoneal injection and aspirin by gavage, and mice in the control group were treated with saline by intraperitoneal injection and water by gavage. Only the administering experimenter was aware of the group allocation. The tumor fragments were transplanted on day 0, and after an intraperitoneal injection of XenoLight D-Luciferin Potassium Salt (Perkin Elmer, Waltham, MA, USA), tumor growth was observed with an IVIS Lumina II (Caliper Life Science, Waltham, MA, USA) beginning on day 8 and then once per week thereafter to obtain in vivo bioluminescence images. The acquired optical data were analyzed using Living Image software. Aspirin was initially administered on day 9 at a dose of 20 mg/kg and then every other day thereafter, while CDDP administration was initiated at a dose of 3 mg/kg on day 17 and then every fourth day thereafter. All mice were euthanized via overdose isoflurane inhalation on day 36 and necropsied for the measurement of tumor diameters with Vernier calipers. The tumor volume and relative tumor inhibition rate were calculated using the following formulas:

V = (L ×W²)/2 and R = (V _controlgroup–V _{treatmentgroup})/V _controlgroup.

Immunohistochemistry (IHC)

Tissue sections (4-µm thick) were prepared from formalin-fixed, paraffin-embedded blocks and were immunohistochemically stained with rabbit anti-p53 (1:100; Abcam, Cambridge, UK), rabbit anti-acetylated p53 (K382) (1:100; Abcam, Cambridge, UK), rabbit anti-Ki67 (1:100; Abcam, Cambridge, UK), rabbit anti-caspase 3 (1:100; Cell Signaling Technology), and rabbit anti-cleaved caspase 3 (1:100; Cell Signaling Technology) antibodies. Localization of the target protein was visualized by incubating sections with a freshly prepared 3,3′-diaminobenzidine (DAB) solution for 3 min. PBS was substituted for the primary antibody as the negative control. Three slides from each tissue were independently evaluated by two observers with an Olympus FV500 optical microscope (Olympus, Tokyo, Japan).

Quantitative analysis of images of immunohistochemical staining

The expression of target proteins in tumors harvested from transplanted mice was quantified by determining the relative percentage of the positive-stained area to the selected total tissue area and the average staining intensity normalized to the intensity of the selected stroma with an imaging processor to exclude the influence of the tumor volume. Slides were reviewed under a light microscope at 400 × magnification, and five regions were randomly selected per slide. ImageJ software was used to analyze the percentage of the positive-stained area and the staining intensity in the tumor tissue from every region, and the overall average value across the slides was considered the level of protein expression.

Statistical analysis

All statistical analyses were performed using SPSS software version 22.0. Data are presented as the means ± standard deviations (SD, n ≥3) values. Student’s t-test or one-way ANOVA was adopted to compare group differences, and significance was defined as p < 0.05.

Aspirin inhibits the growth of human EOC cells in vitro

A survival curve based on the results of MTT assays showed that the half-maximal inhibitory concentration (IC50) values of aspirin in the EOC cell lines A2780, Caov-3 and SK-OV-3 were 1.27 (95% CI [0.93–1.72]) mM, 2.05 (95% CI [1.83–2.30]) mM, and 1.54 (95% CI [1.30–21.82]) mM, respectively (Fig. 1A). Thus, we conducted the functional trial in vitro with doses of 100 and 1,000 µM, which were lower than the IC50 in each cell line.

The results of EdU cell proliferation and colony formation assays illustrated that the proliferation and growth of the A2780, Caov-3 and SK-OV-3 cell lines were significantly suppressed by aspirin in a concentration-dependent manner (Figs. 1B–1E and Figs. S2A–S2D). Moreover, as the aspirin concentration increased, the apoptosis rate in the EOC cell lines increased significantly, and the apoptosis rate differed significantly among the different concentrations (Figs. 1F–1G). In addition, the results of wound healing and transwell migration assays showed that the migration of A2780, Caov-3 and SK-OV-3 cells was suppressed by aspirin in a concentration-dependent manner (Figs. S2A–S2D).

Aspirin enhances the CDDP sensitivity of EOC cells

A2780 and SK-OV-3 cells were treated with CDDP alone or in combination with aspirin to investigate the role of aspirin in mediating CDDP sensitivity in vitro. Cell proliferation (Figs. 2A–2B and Fig. S3A) and colony formation (Figs. 2C–2D and Fig. S2B) were significantly inhibited and cell apoptosis (Figs. 2E–2F and Fig. S2C) was induced by CDDP alone in A2780 and SK-OV-3 cells compared to the corresponding control cells. Moreover, CDDP plus aspirin exerted a significantly stronger effect than CDDP alone.

Orthotopic xenograft mouse models of EOC were established with A2780-Luciferase cells, and tumor bioluminescence signals were visualized in situ to investigate the role of aspirin in mediating CDDP sensitivity in vivo (Fig. 3A). As shown in Fig. 3B, the tumor growth curve of the CDDP plus aspirin group was shallower than that of the CDDP alone group, and the CDDP alone group had a shallower curve than the control group. Moreover, the difference in the tumor volume among the three groups was consistent with the tumor growth curves (Figs. 3C–3D). The tumor inhibition rates in the CDDP group and the CDDP plus aspirin group were 49.3% and 78.4%, respectively.

In addition, the level of caspase 3 in the tumor tissue was not different among the three groups (control, CDDP, and CDDP plus aspirin); however, the level of cleaved caspase 3 was higher in the CDDP group than in the control group and was higher in the CDDP plus aspirin group than in the CDDP group (Fig. 3E). Ki67 expression was reduced in the CDDP group compared to the control group and was lower in the CDDP plus aspirin group than in the CDDP group (Fig. 3E).

Aspirin acetylates p53 in EOC cells and subsequently activates p53-mediated downregulation of target genes

The expression of p53 was not different among the three groups (control, CDDP, and CDDP plus aspirin), but the acetylation level of p53 in the CDDP plus aspirin group was higher than that in both the control group and the CDDP group (Figs. 4A–4B). As shown in Fig. 4C, the level of the p53 protein in the A2780 cell line remained unchanged after treatment with different concentrations of aspirin. However, as the aspirin concentration increased, the acetylation level of p53 gradually increased. Moreover, the mRNA expression levels of the p53 target genes CDKN1A, BAX, FOXF1, PUMA, and RRAD increased significantly in a concentration-dependent manner, as illustrated in Fig. 4D.