Effects of selective cyclooxygenase-2 inhibitor robenacoxib on primary cells derived from feline injection-site sarcoma

Abstract

Feline injection-site sarcomas (FISSs) are highly invasive malignant mesenchymal neoplasms that arise from injection sites in cats. Although the tumorigenesis of FISSs is still uncertain, there is a consensus that FISS is associated with chronic inflammation caused by irritation of injection-related trauma and foreign chemical substances. Chronic inflammation can provide a proper microenvironment for tumour development, which has been known as one of the risk factors of tumorigenesis in many tumours. To investigate the tumorigenesis of FISS and screen for its potential therapeutic targets, cyclooxygenase-2 (COX-2), an inflammation-enhancing enzyme, was selected as a target for this study. In vitro experiments using FISS- and normal tissue-derived primary cells and robenacoxib, a highly selective COX-2 inhibitor, were performed. The results demonstrated that expression of COX-2 could be detected in formalin-fixed and paraffin-embedded FISS tissues and FISS-derived primary cells. Cell viability, migration and colony formation of FISS-derived primary cells were inhibited, and cell apoptosis was enhanced by robenacoxib in a dose-dependent manner. However, susceptibility to robenacoxib varied in different lines of FISS primary cells and was not completely correlated with COX-2 expression. Our results suggest that COX-2 inhibitors could be potential adjuvant therapeutics against FISSs.

Keywords: cyclooxygenase-2; feline injection-site sarcoma; primary cells; robenacoxib.

FIGURE 1

Expression level of COX‐2, vimentin, desmin and α‐SMA in FISS‐7, FISS‐12 and FM primary cells. Negative controls are stained with phosphate‐buffered saline as primary antibody. Length of the scale bars is 50 μm.

FIGURE 2

Cyclooxygenase‐2 expression in feline injection site sarcomas (FISSs) by immunohistochemical staining. Results of FISS‐7 and FISS‐12 are displayed in (A) and (B), respectively. Insets showing higher magnifications of positive cells (400×). (C) Negative control was stained with phosphate‐buffered saline and the result is shown. (D) The macula densa in normal canine kidney is exhibited as positive control. The scale bars showed in (A–C) and (D) are 200 and 50 μm, respectively.

FIGURE 3

Result of western blotting against COX‐2 and beta Actin antibody in FISS and FM cells. (A) Back arrows indicate respective target proteins. (B) The quantified COX‐2 expression levels in different primary cells. The ratio of each primary cell is the fold change of COX‐2 expression when compared to beta Actin expression. No statistical difference is shown between the cell lines.

FIGURE 4

Results of wound healing assay in FISS and FM cells under different concentrations of robenacoxib. (A) Figures illustrate cell migration of FISS‐12 at different concentrations and time points. Widths of the wounds were photographed and measured before (T0) and 24 h (T1) after being treated with different concentrations of robenacoxib. Treatment with 0 μM robenacoxib is considered as a vehicle (DMSO) control, in which the culture medium is only supplemented with 0.5% DMSO. (B) Values of T1 are expressed as the percentages of the wound widths at T0. * Values are statistically significant when compared to the medium control (p < 0.05).

FIGURE 5

Results of clonogenic assay in different lines of primary cells treated with different concentrations of robenacoxib. (A) Graphs of the cell clonogenesis of each line of primary cells on the six‐well plate. (B) Survival fraction of each primary cell line treated with different concentrations of robenacoxib are illustrated. Group treated with 0 μM robenacoxib is designated as vehicle (DMSO) control group. Group treated with medium only is designated as control group. * Represents significant statistical difference than the control group (p < 0.05).

FIGURE 6

Result of TUNEL assay for evaluating cell apoptosis in different lines of primary cells treated with different concentrations of robenacoxib. (A) Apoptotic cells detected by TUNEL assay in each line of primary cells after treatment with different concentrations of robenacoxib. Green spots are positive signals of TUNEL staining. Blue spots are the cell nuclei stained with DAPI as counterstain. Double‐positive cells of TUNEL and DAPI staining highlighted as light blue are interpreted as the apoptotic cells. (B) Result of apoptotic rate expressed as percentage of apoptotic cell count to total cell count. Group treated with 0 μM of robenacoxib is designated as the vehicle (DMSO) control group. Group treated with medium only is designated as the control group. * Represents significant statistical difference than the control group (p < 0.05).