Synergistic growth inhibitory effect of deracoxib with doxorubicin against a canine mammary tumor cell line, CMT-U27

Abstract

Cyclooxygenase (COX) inhibitors have been shown to exert anti-angiogenic and anti-tumor activities on many types of malignant tumors. These anticancer properties make it worthwhile to examine the possible benefit of combining COX inhibitors with other anti-cancer agents. In the present study, we evaluated the potential of deracoxib (DER) in potentiating antitumor activity of doxorubicin (DOX) in canine mammary carcinoma cells (CMT-U27). DER (50-250 µM) enhanced the antiproliferative activity of DOX by reducing the IC50 (approximately 3- to 3.5 fold). Interaction analysis of the data showed that combinations of DOX at 0.9 µM with DER (100-250 µM) produced synergism in the CMT-U27 cell line, with a ratio index ranging from 1.98 to 2.33. In additional studies identifying the mechanism of observed synergistic effect, we found that DER strongly potentiated DOX-caused G0/G1 arrest in cell cycle progression. Also, DER (100-250 µM) augmented apoptosis induction with approximately 1.35- and 1.37- fold increases in apoptotic response caused by DOX in the cells. DER enhanced the antiproliferative effect of DOX in conjunction with induction of apoptosis by modulation of Bcl-2 expression and changes in the cell cycle of the CMT-U27 cell line. Although the exact molecular mechanism of the alterations in the cell cycle and apoptosis observed with DER and DOX combinations require further investigations, the results suggest that the synergistic effect of DOX and DER combinations in CMT therapy may be achieved at relatively lower doses of DOX with lesser side effects. Therefore, combining DER with DOX may prove beneficial in the clinical treatment of canine mammary cancer.

Fig. 1.

Antiproliferative effects of doxorubicin and deracoxib combinations in the CMT-U27 cell line. Cells were treated with the indicated doses of doxorubicin and deracoxib, and cell viability was assayed 72 hr after treatment. Data are expressed as mean percentage of cell viabilities ± standard error (SE). **P<0.01 compared with the doxorubicin-treated group. DOX, doxorubicin; DER, deracoxib.

Fig. 2.

Effects of doxorubicin and deracoxib combination treatment on apoptosis of CMT-U27 cells. (A) The number of viable, early apoptotic, late apoptotic and necrotic cells due to treatment with doxorubicin and deracoxib combinations for 72 hr in the CMT-U27 cell line. The experiment was conducted in three replicates. Data are expressed as the mean ± standard error (SE). *P<0.05 compared with the 0.9 µM doxorubicin-treated group. DOX, doxorubicin; DER, deracoxib. (B) Representative cytograms of the CMT-U27 cell line double stained with Annexin V-FITC and propidium iodide (PI). The numbers written on histograms represent the sum of early and late apoptotic cells (%).

Fig. 3.

Effects of supplementation of PGE₂ on enhancement of growth inhibitory activity of doxorubicin caused by deracoxib in CMT-U27 cells. Cells were treated with or without deracoxib (50-250 µM) in the absence or presence of PGE₂ (1 or 5 µM) and 0.9 µM doxorubicin (A) or 0.09 µM doxorubicin (B) alone or in combination for 72 hr before determination of cell viability by MTT assay. DOX, doxorubicin; DER, deracoxib.

Fig. 4.

Immunocytochemical expression of Bcl-2 in CMT U27 cells. A) Deracoxib 50 µM, moderate immunopositivity; B) Deracoxib 250 µM, slight immunopositivity; C) Doxorubicin 0.09 µM + deracoxib 50 µM, strong immunopositivity; D) Doxorubicin 0.9 µM + deracoxib 250 µM slight immunopositivity; E) Control cells, very strong immunopositivity; F) Negative control cells, no reaction. Bar=20 µm.