Meloxicam decreases the migration and invasion of CF41.Mg canine mammary carcinoma cells

Abstract

Cyclooxygenase (COX)-2 expression is positively correlated with malignant features in canine mammary carcinomas. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity and may therefore possess anticancer effects. Meloxicam is an NSAID that is widely used in human and veterinary medicine. High concentrations of meloxicam have been reported to be antitumorigenic in vitro; however, the effect of meloxicam at concentrations that are equivalent to those that can be obtained in vivo remains unknown. In the current study, the in vitro effects of low-dose meloxicam (0.25 µg/ml) on CF41.Mg canine mammary carcinoma cells were evaluated. The effects on cell proliferation, apoptosis, cell migration and invasion, in addition to the expression of different molecules associated with tumor invasiveness were analyzed. No effect on cell viability and apoptosis were observed. However, cell migration and invasion were significantly reduced following treatment with meloxicam. MMP-2 expression and activity were similarly reduced, explaining the impaired cell invasion. In addition, β-catenin expression was downregulated, while its phosphorylation increased. These results indicate that 0.25 µg/ml meloxicam reduces cell migration and invasion, in part through modulating MMP-2 and β-catenin expression. Additional studies are required to elucidate the mechanism associated with the anti-invasive effect of meloxicam on CF41.Mg cells. The results of the present study suggest that meloxicam has a potential adjunctive therapeutic application, which could be useful in controlling the invasion and metastasis of canine mammary carcinomas.

Keywords: canine mammary carcinoma; cell invasion; cyclooxygenase-2; meloxicam.

Figure 1.

Expression of COX-2 in MDCK and CF41.Mg cells. (A) Representative immunofluorescence images of the (left panel) negative control, where the nuclei were visualized by DAPI, and COX-2 expression in MDCK and CF41.Mg cells respectively (central and right panel). Scale bar, 32 µm. (B) Representative Western blot of the expression of COX-2 in cell lysates, demonstrating a band at 72 kDa in both cell lines. Results are representative of 3 independent experiments. COX, cycloooxgenase; MDCK, Madin-Darby Canine Kidney.

Figure 2.

Cell viability following incubation with different concentrations of meloxicam and/or doxorubicin. (A) CF41.Mg and (B) MDCK cells were incubated for 24 and 48 h with meloxicam (0–25 µg/ml). The percentage of viable cells was determined by MTS assay. Control cells were treated with DMSO alone. (C) Viability of CF41.Mg cells incubated for 24 and 48 h with meloxicam (0–25 µg/ml) and 500 ng/ml doxorubicin. (D) CF41.Mg cells were incubated with meloxicam (0–1 µg/ml) and 24 h later 500 ng/ml doxorubicin was added. Cell viability was measured at 24 and 48 h. Values are presented as the mean ± standard deviation of ≥3 independent experiments performed in triplicate. *P<0.05.

Figure 3.

Meloxicam (0.25 µg/ml) has no effect on CF41.Mg cell apoptosis. Doxorubicin alone (500 ng/ml) was used as a positive control. A total of 3 independent experiments were performed and values are presented as the mean ± standard deviation. Flow cytometry plots and analysis of the data are illustrated. *P<0.05 vs. the control group.

Figure 4.

Meloxicam (0.25 µg/ml) decreases the migration of CF41.Mg cells. Incubation with 0.25 µg/ml meloxicam for 24 and 48 h inhibited the migration of cells compared with the DMSO-treated control cells in a wound healing assay. A total of 6 repeats were performed and results are presented as the mean ± standard deviation. Representative images of the wound healing assay and quantification if the results are presented. Scale bar, 90 µm. *P<0.05 vs. the control group.

Figure 5.

Meloxicam (0.25 µg/ml) decreases the invasion of CF41.Mg cells. The invasive cells were counted in 3 independent Matrigel invasion experiments and the results are presented as the mean number of invading cells ± standard deviation. Representative microphotographs and a graph of the results are presented. Scale bar, 64 µm. *P<0.05 vs. the control group.

Figure 6.

Meloxicam (0.25 µg/ml) increases the activity and expression of MMP-2 in CF41.Mg cells. (A) Representative gelatin zymography image of MMP-2 and 9. (B) Representative Western blot and respective histogram showing the expression of MMP-2. Values are expressed as mean ± SD and at least three independent experiments were performed. *P<0.05 vs. the control group. M24, meloxicam treatment for 24 h; M48, meloxicam treatment for 48 h.

Figure 7.

Meloxicam (0.25 µg/ml) increases β-catenin expression in CF41.Mg cells. Representative western blots and quantification of (A) total β-catenin and (B) p-β-catenin. Values are presented as the mean ± standard deviation of ≥3 independent experiments. *P<0.05 vs. the control group. p, phosphorylated; M24, meloxicam treatment for 24 h; M48, meloxicam treatment for 48 h.

Figure 8.

Meloxicam (0.25 µg/ml) does not affect COX-2 expression in CF41.Mg cells. (A) Representative western blots showing the expression of COX-2 in CF41.Mg cells for 24 (left blot) and 48 h (right blot) and (B) quantification of COX-2 expression. Values are presented as the mean ± standard deviation of 3 independent experiments.