5-lipoxygenase expression and tepoxalin-induced cell death in squamous cell carcinomas in cats

Abstract

Objective: To assess expression pattern and subcellular compartmentalization of 5-lipoxygenase in cutaneous, UV radiation-induced, and oral squamous cell carcinomas (SCCs) in cats and determine the effects of cyclooxygenase or 5-lipoxygenase inhibition on proliferation or apoptosis in a feline oral squamous cell carcinoma (SCCF1) cell line.

Sample: 60 archived paraffin-embedded samples of SCCs from 60 cats and SCCF1 cells.

Procedures: Retrospective immunohistochemical analysis of the archived samples of SCCs (20 cutaneous, 20 UV radiation-induced, and 20 oral tumors) was performed. Cell culture proliferation assays involving SCCF1 cells were performed, and tepoxalin-induced apoptosis and signaling were examined via western blotting and annexin V staining.

Results: Immunohistochemically, staining for 5-lipoxygenase was most frequently of greatest intensity in oral SCCs, whereas staining of cutaneous and UV radiation-induced lesions had less consistent 5-lipoxygenase expression. Exposure of SCCF1 cells to the 5-lipoxygenase inhibitor tepoxalin resulted in apoptosis; the effect appeared to be mediated via alteration of cell signaling rather than via suppression of lipid mediators that are typically produced as a result of 5-lipoxygenase activity.

Conclusions and clinical relevance: In cats, expression of 5-lipoxygenase in SCCs appeared to differ depending on tumor location. The influence of tepoxalin-induced 5-lipoxygenase inhibition on a 5-lipoxygenase-expressing cell line coupled with the notable expression of 5-lipoxygenase in oral SCCs suggested that 5-lipoxygenase inhibition may have therapeutic benefits in affected cats. Although the safety of tepoxalin in cats has yet to be investigated, 5-lipoxygenase inhibitors should be evaluated for use as a potential treatment for SCCs in that species.

Figure 1—

Representative photomicrographs of sections of oral or UV radiation–induced SCCs in 3 cats to illustrate results of immunohistochemical staining for 5-lipoxygenase. Sections from each were processed with anti–5-lipoxygenase rabbit polyclonal rabbit antibody or a negative control rabbit antibody, and intensity and subcellular compartmentalization of 5-lipoxygenase–specific staining were assessed. Staining intensity was subjectively scored as follows: 1 = weak staining intensity, 2 = moderate staining intensity, and 3 = marked staining intensity. Subcellular compartmental staining was categorized as cytoplasmic, perinuclear, or nuclear, which were each further categorized as focal if < 10% of neoplastic cells were stained, patchy if 11% to 50% of cells were stained, and diffuse if > 50% of cells were stained. A—Section of an oral SCC that underwent immunohistochemical staining with anti–5-lipoxygenase antibody.The staining intensity score for this tumor is 3, and there is uniform cytoplasmic staining and nuclear staining. Bar = 200 μm. B—Section of another oral SCC that underwent immunohistochemical staining with anti–5-lipoxygenase antibody. Notice the intense cytoplasmic staining and patchy nuclear staining of variable intensity (arrows). Bar = 50 μm. C—Section of a UV radiation–induced SCC that underwent immunohistochemical staining with anti–5-lipoxygenase antibody. Notice the intense cytoplasmic 5-lipoxygenase–specific staining and patchy punctuate perinuclear staining (arrows). Bar = 50 μm. D—Section of the same oral SCC in panel A that underwent immunohistochemical staining with control rabbit antibody. Bar = 200 μm.

Figure 2—

Representative results of western blot analysis for 5-lipoxygenase in SCCF1 and A-72 thymic fibroblast lysates, compared with 2 μg of recombinant his-5-lipoxygenase (rhis–5-LOX) and mean ± SD viability of SCCF1 cells treated for 48 hours with various concentrations of piroxicam, TAM, licofelone, tepoxalin, and tepoxalin with TAM, or a vehicle control (DMSO). For the western blot analysis, 30 μg of each lysate was loaded into each lane. Cell viability was determined by use of MTT assays, and results are expressed as a percentage of vehicle control–treated cell viability. *At this concentration, percentage viability of tepoxalin-treated and tepoxalin-TAM–treated cells differed significantly (P < 0.05) from the findings for the vehicle control–treated cells. †At this concentration, percentage viability of licofelone-treated cells differed significantly (P < 0.05) from the findings for the vehicle control–treated cells.

Figure 3—

Growth curves for SCCF1 cells in the presence of various concentrations of tepoxalin or a vehicle control (DMSO) by use of MTT assays. Optical density readings at a wavelength of 540 nm were obtained via spectophotometry at day 0 (prior to exposure to tepoxalin) and every other day thereafter during serial dilution treatment with tepoxalin or vehicle control. At day 6, all concentrations of tepoxalin (0.625 to 10μM) resulted in significant (P < 0.05) decreases in cell proliferation, compared with findings for vehicle control–treated cells.

Figure 4—

Evidence supporting the apoptotic effects of tepoxalin on SCCF1 cells in culture. A—Results of western blot analysis for markers of apoptosis in SCCF1 cells that were treated with tepoxalin (10μM) or vehicle control (DMSO) for 48 hours, with β-actin as a marker of equal loading. Samples of cells were lysed and were assessed before treatment (0 hours) and at 12, 24, 36, and 48 hours, and representative blots are illustrated. There is late activation of caspase-3 (Act caspase 3) and diminished Akt serine 473 phosphorylation (Ser473-pAkt) prior to caspase activation. As well as less phosphorylation of Akt, the amount of total Akt decreases with time. The amount of death receptor 5 (DR 5) is stable until apoptosis occurs at 36 to 48 hours. B—Photomicrographs (obtained by use of a fluorescence microscope) of preparations of SCCF1 cells that were treated with tepoxalin (10μM; right panel) or vehicle control (DMSO; left panel) for 48 hours and stained with annexin V Alexa 488 and 7-AAD. Annexin V (positive green) and 7-AAD (positive red) staining reveals apoptotic (annexin V–negative and 7-AAD–negative) and subsequent necrotic (annexin V–positive and 7-AAD–positive) cells treated with tepoxalin (right panel) or vehicle control (left panel). C—Graphs depicting mean ± SD percentages of apoptotic (left panel) or necrotic (right panel) SCCF1 cells after 24, 36, and 48 hours of exposure to tepoxalin or the vehicle control (DMSO). *At this time point, the value for tepoxalin-treated cells is significantly (P < 0.01) different from the value for vehicle control–treated cells.