Parecoxib and 5-Fluorouracil Synergistically Inhibit EMT and Subsequent Metastasis in Colorectal Cancer by Targeting PI3K/Akt/NF-κB Signaling

Abstract

Colorectal cancer is one of the most common causes of cancer mortality worldwide, and innovative drugs for the treatment of colorectal cancer are continually being developed. 5-Fluorouracil (5-FU) is a common clinical chemotherapeutic drug. Acquired resistance to 5-FU is a clinical challenge in colorectal cancer treatment. Parecoxib is a selective COX-2-specific inhibitor that was demonstrated to inhibit metastasis in colorectal cancers in our previous study. This study aimed to investigate the synergistic antimetastatic activities of parecoxib to 5-FU in human colorectal cancer cells and determine the underlying mechanisms. Parecoxib and 5-FU synergistically suppressed metastasis in colorectal cancer cells. Treatment with the parecoxib/5-FU combination induced an increase in E-cadherin and decrease in β-catenin expression. The parecoxib/5-FU combination inhibited MMP-9 activity, and the NF-κB pathway was suppressed as well. Mechanistic analysis denoted that the parecoxib/5-FU combination hindered the essential molecules of the PI3K/Akt route to obstruct metastatic colorectal cancer. Furthermore, the parecoxib/5-FU combination could inhibit reactive oxygen species. Our work showed the antimetastatic capacity of the parecoxib/5-FU combination for treating colorectal cancers via the targeting of the PI3K/Akt/NF-κB pathway.

Keywords: 5-fluorouracil; PI3K/Akt pathway; colorectal cancer; metastasis; parecoxib; reactive oxygen species.

Figure 1

Effect of parecoxib and 5-FU on cell viability, as assessed by MTT assay. After incubation, cell viability was assessed by MTT analysis. Significant differences in the untreated group (UN) are shown as follows: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Figure 2

Effect of parecoxib and 5-FU on cell migration and invasion by transwell and matrix gel assays in DLD-1 cells. (A,B) Migration assay. (D,E) Invasion assay. (A,D) Arbitrary fields from each of the triplicate migration assays were calculated using a phase-contrast microscope (magnification 200×). (B,E) The absorbance of crystal violet was determined at 570 nm by a microplate reader. The values are displayed as the mean ± SD of separate trials. Significant differences are set at p < 0.001 (***). (C) Isobologram analysis of the parecoxib and 5-FU combination in DLD-1 cells. The trials were conducted at least three times. A descriptive trial is shown.

Figure 3

(A) After treatment, the cells that migrated to the wounded regions were calculated by a phase-contrast microscope (magnification 200×). (B) Percentages of DLD-1 cells that migrated to the wound area following treatment were evaluated. Significant differences in the untreated group (UN) are denoted as p < 0.001 (***). The * is a statistical symbol. Significant differences in parecoxib (3 μM) alone or 5-FU (20 μM) alone are indicated as p < 0.001 (###).

Figure 4

Effect of parecoxib and 5-FU on EMT. After treatment, the levels of protein expression were evaluated using the extracted proteins and assessed by Western blot. Actin, tubulin, or GAPDH were used as internal controls.

Figure 5

Effect of parecoxib and 5-FU on MMP-9 activity. After treatment, the conditional media were used on non-reduced denatured 12% polyacrylamide gel containing gelatin and stained with Coomassie Blue.

Figure 6

Effect of parecoxib and 5-FU on the p-Akt and NF-κB pathways. The cells were treated with drugs for (A) 24 and 48 h, and for (B) 1 h. After incubation, levels of protein expression were assessed via Western blot analysis. Actin, tubulin, or GAPDH were selected as loading controls.

Figure 7

Effect of overexpression of Akt phosphorylation in parecoxib- and 5-FU-treated DLD-1 cells. (A,D) Phosphorylation of Akt was detected by Western blot. GAPDH and actin were selected as loading controls. (B,E) Random areas from each of the triplicate migration assays were assessed using a phase-contrast microscope (magnification 200×). (C,F) The absorbance of crystal violet was detected at 570 nm by using a microplate reader. The data are shown as the mean ± SD of separate tests. Significant differences are expressed as p < 0.001 (***).

Figure 8

Effect of parecoxib and 5-FU on intracellular ROS in DLD-1 cells. After treatment, all cells were incubated with DCFH-DA for intracellular ROS detection and assessed using a flow cytometer. The data in each panel show the mean fluorescence intensity of DCF inside the cells. The data are shown as the mean ± SD (n = 5–8) of individual experiments.

Figure 9

Effect of parecoxib and 5-FU on cell migration and invasion by transwell and matrix gel assays in SW480 cells. (A,B) Migration assay. (D,E) Invasion assay. (A,D) Random fields from each of the triplicate migration assays were calculated by a phase-contrast microscope (magnification 200×). (B,E) The absorbance of crystal violet was determined at 570 nm by using a microplate reader. The values are displayed as the mean ± SD of separate trials. Significant differences are expressed as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). (C) Isobologram analysis of the parecoxib and 5-FU combination in SW480 cells. The trials were conducted at least three times. A descriptive trial is shown.