Firocoxib as a Potential Neoadjuvant Treatment in Canine Patients with Triple-Negative Mammary Gland Tumors

Abstract

This study aimed to investigate the pro-apoptotic effects of NSAID (Previcox^®) in vitro and in vivo. Two CMT cell lines, one from the primary tumor and one from bone metastasis, were treated with firocoxib and MTT assay was performed to determine the half-maximal inhibitory concentration (IC₅₀) value. The firocoxib IC₅₀ for the cell lines UNESP-CM5 and UNESP-MM1 were 25.21 µM and 27.41 µM, respectively. The cell lines were then treated with the respective firocoxib IC₅₀ concentrations and annexin V/propidium iodide (PI) assay was performed, to detect the induction of apoptosis in both cells (Annexin+/PI+). We conducted an in vivo study involving female dogs affected by CMT and divided them into control and treatment groups. For both groups, a biopsy was performed on day 0 (D0) and a mastectomy was performed on day 14 (D14). In the treatment group, after biopsy on D0, the patients received Previcox^® 5 mg/kg PO once a day until mastectomy was performed on D14. COX-2/caspase-3 double immunostaining was performed on samples from D0 and D14, revealing no difference in the control group. In contrast, in the treatment group Previcox^® increased the number of COX-2 positive apoptotic cells. Therefore, firocoxib can induce apoptosis in CMT cells in vitro and in vivo, and Previcox^® can be a potential neoadjuvant treatment for patients with mammary cancer.

Keywords: caspase-3; chemotherapy; cyclooxygenase-2; nonsteroidal anti-inflammatory drug.

Figure 1

COX-2 expression and morphological characteristics of UNESP-CM5 and UNESP-MM1 from tumor samples and cancer cell lines. (A): primary tumor that caused the UNESP-CM5 cancer cell line presenting COX-2 positive immunofluorescence (red color). (B): tumor sample that caused UNESP-MM1 cell line presenting COX-2 overexpression (red color) by immunofluorescence. (C): fibroblastoid to polygonal morphology of UNESP-CM5 cell line (20×). (D): polygonal morphology of UNESP-MM1 cell line (20×).

Figure 2

Cell culture experiments of UNESP-CM5 and UNESP-MM1 cancer cell lines. (A): cellular viability of canine UNESP-CM5 cell line under different firocoxib concentrations. (B): UNESP-MM1 cell line viability after treatment with different firocoxib concentrations. (C): Annexin V (AN) and propidium iodide (PI) assay in a pool of both cell lines (UNESP-CM5 and UNESP-MM1) without treatment as a negative control. The control cells presented more than 85% viability (PI−/AN−). (D): UNESP-CM5 cell line presenting 33.1% of the cells with PI+/AN+ and 45.9% of the cells negative for both. (E): UNESP-MM1 cell line showing more than 74% of the cells positive for PI and AN (PI+/AN+). The asterisk (*) means statistical difference compared with control group.

Figure 3

Double immunostaining of COX-2 (cytoplasmic staining—brown) and cleaved caspase-3 (nuclear staining—red) in canine mammary gland tumor samples. (A,B): COX-2/caspase-3 double immunostaining at D0. (C): COX-2/caspase-3 double staining of the control group tissue samples, on D14. No difference was found when the tissue samples from patients on D0 were compared with those on D14. (D): increased number of double-stained cells in a sample on D14, compared with those on D0. Harris hematoxylin counterstain, 3 3′-diaminobenzidine chromogen, and 20× magnification.