Aspirin Blocks EGF-stimulated Cell Viability in a COX-1 Dependent Manner in Ovarian Cancer Cells

Abstract

Objective: Although aspirin has been associated with a reduction of the risk of cancer when used as a nonsteroidal anti-inflammatory drug, its use to reduce the risk of ovarian cancer is controversial. Ovarian cancer cells usually express high levels of cyclooxygenase-1 (COX)-1. Because aspirin is a rather selective inhibitor of COX-1, the ability of aspirin to reduce the risk of ovarian cancer may be dependent on the level of COX-1 expression in those cells. Furthermore, epidermal growth factor receptor (EGFR) is frequently overexpressed in the malignant phenotype of ovarian cancer leading to increased cell proliferation and survival. Here we investigated if aspirin attenuates EGFR-activated ovarian cancer cell growth in a COX-1 dependent manner.

Methods: Cell viability assays and Western blot analyses were used to determine the effect of aspirin on EGF-stimulated cell proliferation. Gene silencing and gene expression techniques were employed to knockdown or to express COX-1, respectively.

Results: Aspirin inhibited cell viability induced by EGF in a dose dependent manner in COX-1 positive ovarian cancer cells. On the other hand, aspirin had no effect on cell viability in COX-1 negative ovarian cancer cells. In particular, aspirin decreased phosphorylated Akt and Erk activated by EGF. COX-1 silencing in COX-1 positive cells attenuated the inhibitory effect of aspirin on EGF-stimulated cell viability. Furthermore, we developed a COX-1 expressing cell line (SKCOX-1) by stably transfecting COX-1 expression vector into COX-1 negative SKOV-3 cells. SKCOX-1 cells were more responsive to aspirin when compared to cells transfected with empty vector, and decreased EGF-activated Akt and Erk as well as cell viability.

Conclusions: Taken together, aspirin inhibits viability of ovarian cancer cells by blocking phosphorylation of Akt and Erk activated by EGF. Thus it may potentiate the therapeutic efficacy of drugs used to treat COX-1 positive ovarian cancer subsets.

Keywords: Akt; COX-1; EGF; Erk; aspirin; cell viability.; ovarian cancer.

Fig 1

Aspirin differentially regulates cell viability in COX-1 positive versus negative ovarian cancer cells. (A) Effects of aspirin on cellular proliferation in ovarian cancer cell lines OVCAR-3, SKOV-3, A2780 and TOV-21G. Cells were treated for 48 hours with aspirin (0.25, 0.5 and 1 mmol/L) in a dose-dependent manner. The cell viability assay was performed by using MTT, and values were normalized to untreated controls. Experiments were performed in triplicate and all data are shown as mean ± SE. # Indicates a significant decrease (p≤0.05) using ANOVA and Tukey's pairwise analyses. (B) COX-1 and COX-2 protein expressions were evaluated in ovarian cancer cell lines by western blot. β-actin was used as a loading control. As a positive control of COX-2, SW480 cells were used at 30 minute TNF (10 ng/ml) post-treatment.

Fig 2

ASA decreases EGFR-activated cell viability by blocking phosphorylation of Erk and Akt. (A) A dose-dependant effect of EGF on cell viability in COX-1 positive OVCAR-3 cells. Cells were treated for 48 hours with EGF (0-40 ng/ml). *, ** Indicates a significant increase (p≤0.05) using ANOVA and Tukey's pairwise analyses. (B) Effects of aspirin on EGFR-activated cell viability in COX-1 positive OVCAR-3 cells. Cells were treated for 48 hours with aspirin (0.25, 0.5 and 1 mmol/L) in the absence or presence of EGF (10 ng/ml). The cell proliferation assay was performed using MTT, and values were normalized to untreated controls. Experiments were performed in triplicate and all data are shown as means ± SE. *, # Indicate a significant increase or decrease (p≤0.05), respectively, by Student's-t test. The inhibitory effect of aspirin on EGFR, Akt (C) Erk (D) activation in OVCAR-3 cells by Western blot. Cells were pretreated with aspirin (1 mmol/L) for 24 hours followed by EGF (10 ng/ml) treatment for 0-120 minutes as shown. pEGFR (tyr1068), pAkt, pErk, pp38 and pSAPK/JNK indicate phosphorylated EGFR, Akt, Erk, p38, and SAPK/JNK, respectively. β-Actin was used as a loading control. As positive controls for phosphorylated p38 and SAPK/JNK, SW480 cells were used at 30 minute TNF (10 ng/ml) post-treatment.

Fig 3

Silencing COX-1 with a small interfering RNA blocks inhibitory effect of aspirin on cell viability in OVCAR-3 cells. (A) Confirmation of COX-1 knockdown in OVCAR-3 cells by using COX-1 siRNA. Whole cell lysates were prepared and a western blot was carried out using COX-1 specific antibody. β-Actin was used as a loading control. (B) Effects of COX-1 siRNA on the inhibitory effect of aspirin on basal and EGF-stimulated cell viability in OVCAR-3 cells. Cells were transiently transfected with Control or COX-1 siRNAs (final concentration 10 nmol/L) for 48 hours followed by treatment for 48 hours with EGF (10 ng/mL). A cell viability assay was performed using MTT and values were normalized to untreated controls. Experiments were performed in triplicate and all data are shown as means ± SE. *, # Indicate a significant increase or decrease (p≤0.05), respectively, by Student's-t test.

Fig 4

COX-1 contributes to cell viability in ovarian cancer cells. (A) Confirmation of COX-1 expression in COX-1 stable (SKCOX1) transfected cells. Whole cell lysates were prepared from each cell line and a western blot was carried out using a COX-1 specific antibody. β-Actin was used as a loading control; OVCAR-3 cells served as COX-1 positive cell line control. (B) Comparison of cell viability in COX-1 null SKpcDNA and positive SKCOX-1 cells. Experiments were performed in triplicate and all data are shown as means ± SE. * Indicates a significant (p≤0.05) increase in SKCOX-1 cells compared to SKpcDNA cells when a Student's-t test was made.

Fig 5

ASA blocks EGFR-activated cell viability by blocking phosphorylation of Erk and Akt in COX-1 expressing cells. Effects of aspirin on EGFR-activated cell viability in SKpcDNA (A) and SKCOX-1 cells (B). Cells were treated for 48 hours with aspirin (1 mmol/L) in the absence or presence of EGF (10 ng/ml). The cell viability assay was performed by using MTT, and values were normalized to untreated controls. Experiments were performed in triplicate and all data are shown as means ± SE. *, # Indicate a significant increase or decrease (p≤0.05), respectively, by Student's-t test. The inhibitory effect of aspirin on EGFR, Erk and Akt activation in SKpcDNA (C) and SKCOX-1 cells (D) by western blot analysis. Cells were pretreated with aspirin (1 mmol/L) for 24 hours followed by the addition of EGF (10 ng/ml) for 0-120 minutes. pEGFR, pErk and pAkt indicate phosphorylated EGFR, Erk and Akt, respectively. β-Actin was used as a loading control.