Aspirin exerts high anti-cancer activity in PIK3CA-mutant colon cancer cells

Abstract

Evidence suggests that nonsteroidal anti-inflammatory drug aspirin (acetylsalicylic acid) may improve patient survival in PIK3CA-mutant colorectal carcinoma, but not in PIK3CA-wild-type carcinoma. However, whether aspirin directly influences the viability of PIK3CA-mutant colon cancer cells is poorly understood. We conducted in vitro experiments to test our hypothesis that the anti-proliferative activity of aspirin might be stronger for PIK3CA-mutant colon cancer cells than for PIK3CA-wild-type colon cancer cells. We measured the anti-proliferative effect of aspirin at physiologic concentrations in seven PIK3CA-mutant and six PIK3CA-wild-type human colon cancer cell lines. After exposure to aspirin, the apoptotic index and cell cycle phase of colon cancer cells were assessed. In addition, the effect of aspirin was examined in parental SW48 cells and SW48 cell clones with individual knock-in PIK3CA mutations of either c.3140A>G (p.H1047R) or c.1633G>A (p.E545K). Aspirin induced greater dose-dependent loss of cell viability in PIK3CA-mutant cells than in PIK3CA-wild-type cells after treatment for 48 and 72 hours. Aspirin treatment also led to higher proportions of apoptotic cells and G0/G1 phase arrest in PIK3CA-mutant cells than in PIK3CA-wild-type cells. Aspirin treatment of isogenic SW48 cells carrying a PIK3CA mutation, either c.3140A>G (p.H1047R) or c.1633G>A (p. E545K), resulted in a more significant loss of cell viability compared to wild-type controls. Our findings indicate that aspirin causes cell cycle arrest, induces apoptosis, and leads to loss of cell viability more profoundly in PIK3CA-mutated colon cancer cells than in PIK3CA-wild-type colon cancer cells. These findings support the use of aspirin to treat patients with PIK3CA-mutant colon cancer.

Keywords: NSAID; PI3K; anti-tumor effect; colorectal cancer; isogenic cell model.

Figure 1. Aspirin causes decreased cell viability in PIK3CA-mutant human colon cancer cells

(A) Dose-response curves of PIK3CA-mutant (blue lines) or PIK3CA-wild-type (red lines) human colon cancer cells after treatment with increasing concentrations of aspirin (0, 0.5, 1, 2, 4, 6, 8, 10 and 12 mM) for 48 and 72 hours. (B) Dose-response curves of human colon cancer cells with BRAF-mutant (blue lines) or BRAF-wild-type (red lines) as well as KRAS-mutant (blue lines) or KRAS-wild-type (red lines) after treatment with increasing concentrations of aspirin (0, 0.5, 1, 2, 4, 6, 8, 10 and 12 mM) for 48 hours. Percent cell viability is relative to that of DMSO-treated control cells. The data shown represent mean ± standard deviation of three replicates. DMSO, dimethyl sulfoxide; WT, wild-type; MUT, mutation.

Figure 2. Aspirin treatment results in more apoptosis in PIK3CA-mutant human colon cancer cell lines

PIK3CA-mutant human colon cancer cell lines (HCT15, HCT116, and SW948) and PIK3CA-wild-type colorectal cancer cell lines (COLO205, SW480, and SW620) were incubated with aspirin (1, 2.5, and 5 mM) or DMSO for 48 and 72 hours. Apoptosis analysis was performed by flow cytometry with Annexin V-FITC/PI staining. The percentage of Annexin V-FITC/PI positive cells after aspirin treatment at each dose and time point for all cell lines were normalized by DMSO group and compared according PIK3CA status. Student's t-test was performed to determine significance. The data shown represent mean ± standard deviation of three replicates. ^*P value < 0.05. ^**P value < 0.01. DMSO, dimethyl sulfoxide; MUT, mutation; WT, wild-type.

Figure 3. Aspirin leads to a higher proportion of cells in G₀/G₁ phase arrest in PIK3CA-mutant human colon cancer cell lines

PIK3CA-mutant human colon cancer cell lines (HCT116, HCT15, and SW948) and PIK3CA-wild-type colorectal cancer cell lines (COLO205, SW620, and SW480) were incubated with aspirin (0, 1, 2.5, and 5 mM) for 48 and 72 hours. Cell cycle analysis was performed by flow cytometry with PI staining. The percentage of G₀/G₁ phase cells of each aspirin treatment point was normalized by DMSO group and was compared according PIK3CA status. Student's t-test was performed to determine significance. The data shown represent mean ± standard deviation of three replicates. ^*P value < 0.05. ^**P value < 0.01. DMSO, dimethyl sulfoxide; MUT, mutation; WT, wild-type.

Figure 4. Knock-in of PIK3CA mutations sensitizes colon cancer cells to aspirin

Dose-response curves of parental SW48 cells (red) and individual knock-in of PIK3CA-activating mutations at either alleles c.3140A>G (p.H1047R) or c.1633G>A (p.E545K) of SW48 cells (blue) were treated with aspirin (0, 0.5, 1, 2, 4, 6 and 8 mM) for 48 and 72 hours. Percent cell viability is relative to that of DMSO-treated control cells. The data shown represent mean ± standard deviation of three replicates. DMSO, dimethyl sulfoxide; MUT, mutation; WT, wild-type.