In Vitro Assessment of the Synergistic Effect of Aspirin and 5-Fluorouracil in Colorectal Adenocarcinoma Cells

Abstract

Although remarkable progress has been made, colorectal cancer remains a significant global health issue. One of the most challenging aspects of cancer treatment is the resistance of tumor cells to classical chemotherapy. Conventional therapy for colorectal cancer often involves the use of 5-fluorouracil as a chemotherapeutic agent. Aspirin, a drug used primarily to prevent cardiovascular complications, became a focus of attention due to its potential use as an antitumor agent. The purpose of the study was to evaluate the potential synergistic cytotoxic effects of aspirin and 5-fluorouracil on colorectal adenocarcinoma cells. The viability of cells, the impact on the morphology and nuclei of cells, the potential antimigratory effect, and the impact on the expression of the major genes associated with cell apoptosis (Bcl-2, Bax, Bad), as well as caspases 3 and 8, were evaluated. The results indicated that the two compounds exerted a synergistic effect, causing a reduction in cell viability accompanied by changes characteristic of the apoptosis process-the condensation of nuclei and the reorganization of actin filaments in cells, the reduction in the expression of the Bcl-2 gene, and the increase in the expression of Bax and Bad genes, along with caspases 3 and 8. Considering all these findings, it appears that aspirin may be investigated in depth in order to be used in conjunction with 5-fluorouracil to increase antitumor activity.

Keywords: 5-fluorouracil; aspirin; bcl-2 proteins; caspases; colorectal adenocarcinoma; immunofluorescence staining.

Figure 1

In vitro assessment of the cytotoxic effects at the level of HaCaT cells after 72 h of treatment of: (A) acetylsalicylic acid—ASA (1, 2.5, 5, 7.5, and 10 mM) and 5-Fluorouracil—5-FU (5, 10, 25, 50, and 75 μM); and (B) combination of ASA (2.5 mM) and 5-FU (5, 10, 25, 50, and 75 μM). The results are expressed as percentages, based on the standard deviation of three independent experiments. Statistical analysis was conducted using the one-way ANOVA method and Dunnett’s multiple-comparisons post-test (* p < 0.1; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 2

In vitro assessment of the cytotoxic effects at the level of HT-29 cells after 72 h of treatment of: (A) acetylsalicylic acid—ASA (1, 2.5, 5, 7.5, and 10 mM) and 5-Fluorouracil—5-FU (5, 10, 25, 50, and 75 μM); and (B) combination of ASA (2.5 mM) and 5-FU (5, 10, 25, 50, and 75 μM). The results are expressed as percentages, based on the standard deviation of three independent experiments. Statistical analysis was conducted using the one-way ANOVA method and Dunnett’s multiple-comparisons post-test (**** p < 0.0001).

Figure 3

Evaluation of the effects of 72 treatments with ASA, 5-FU, and ASA+5-FU on the morphologies of HaCaT cells. Scale bars indicate 50 µm.

Figure 4

Evaluation of the effects of 72 treatments with ASA, 5-FU, and ASA+5-FU on the morphologies of HT-29 cells. Scale bars indicate 50 µm.

Figure 5

Evaluation of the effects of 72 treatments with ASA, 5-FU, and ASA+5-FU on the structures of nuclei (blue) and F-actin fibers (red) of HaCaT cells. Pictures were taken with the 20× objective, scale bars indicating 50 µm. The yellow arrows indicate the main changes (nuclear condensation, reorganization of actin filaments).

Figure 6

Evaluation of the apoptotic index (AI) of human keratinocytes, HaCaT, after stimulation with ASA 2.5 mM, 5-FU 25 μM, and a combination of the two compounds for a period of 72 h. The results are expressed as percentages and standard deviation as a result of three independent experiments. The statistical analysis involved one-way ANOVA analysis and a Dunnett’s multiple-comparisons post-test (** p < 0.01).

Figure 7

Evaluation of the effects of 72 treatments with ASA, 5-FU, and ASA+5-FU on the structures of nuclei (blue) and F-actin fibers (red) of HT-29 cells. Pictures were taken with the 20x objective, scale bars indicating 50 µm. The yellow arrows indicate the main changes (nuclear condensation, reorganization of actin filaments).

Figure 8

Evaluation of the apoptotic index (AI) of HT-29 cells after stimulation with ASA 2.5 mM, 5-FU 25 μM, and a combination of the two compounds for a period of 72 h. The results are expressed as percentages and standard deviation as a result of three independent experiments. The statistical analysis involved one-way ANOVA analysis and a Dunnett’s multiple-comparisons post-test (* p < 0.1; *** p < 0.001; **** p < 0.0001).

Figure 9

(A) Photographic evaluation of the migration capacity of HaCaT cells after 24 h treatment with ASA (2.5 mM), 5-FU (25 μM), and ASA 2.5 mM + 5-FU 25 μM. The images were taken at the beginning of the experiment (0 h) and at the end (24 h). The scale bars indicate 1000 µm. (B) Quantitative analysis of post-treatment migration capacity. The results are expressed as a percentage as the migration rate after 24 h of treatment and as the standard deviation of three independent experiments. The statistical analysis was carried out by the one-way ANOVA method followed by the Dunett’s multiple-comparisons post-test (** p < 0.01; *** p < 0.001).

Figure 10

(A) Photographic evaluation of the migration capacity of HT-29 cells after 24 h treatment with ASA (2.5 mM), 5-FU (25 μM), and ASA 2.5 mM + 5-FU 25 μM. The images were taken at the beginning of the experiment (0 h) and at the end (24 h). The scale bars indicate 1000 µm. (B) Quantitative analysis of post-treatment migration capacity. The results are expressed as a percentage as the migration rate after 24 h of treatment and as the standard deviation of three independent experiments. The statistical analysis was carried out by the one-way ANOVA method followed by the Dunett’s multiple-comparisons post-test (** p < 0.01; **** p < 0.0001).

Figure 11

Relative fold expression of mRNA expression of pro- and anti-apoptotic mitochondrial markers in HT-29 cells after stimulation with ASA (2.5 mM), 5-FU (25 µM), and ASA 2.5 mM + 5-FU 25 μM for 72 h. The results were reported for 18 s and for the control group (non-stimulated cells) and expressed as mean values ± SD as a result of three independent experiments. The statistical analysis was performed by applying the one-way ANOVA method and Dunnett’s post-test (** p < 0.01, **** p < 0.0001).

Figure 12

Relative fold expression of mRNA expression of caspases 3 and 8 in HT-29 cells after stimulation with ASA (2.5 mM), 5-FU (25 µM), and ASA 2.5 mM + 5-FU 25 μM for 72 h The results were reported for 18 s and for the control group (non-stimulated cells) and expressed as mean values ± SD as a result of three independent experiments. The statistical analysis was performed by applying the one-way ANOVA method and Dunnett’s post-test (** p < 0.01, **** p < 0.0001).