Expression and regulation of cyclooxygenase‐2 in normal and neoplastic canine keratinocytes

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Squamous cell carcinoma (SCC) is one of the most common cancers in dogs, yet relatively little is known about the molecular events involved in its development. Increasing evidence implicates cyclooxygenase-2 (COX-2) in the pathogenesis of various cancers in humans and animals. COX-2 overexpression has recently been demonstrated in canine SCCs. The objective of our study was to characterize the expression and regulation of COX-2 in normal and neoplastic canine keratinocytes (CKs) to provide an in vitro system to investigate the implication of COX-2 in SCC oncogenesis in dogs. Cell lines derived from normal CKs and neoplastic CKs (SCCs) were cultured in the absence or presence of agonists, and immunoblots, immunocytochemistry, radioimmunoassays and a cell proliferation assay were used to characterize COX-2 expression and action. Results showed that neoplastic keratinocytes had a higher basal COX-2 expression than normal keratinocytes. In both cell lines, stimulation with the tumour promoter phorbol 12-myristate 13-acetate induced a time-dependent increase in COX-2 protein, with COX-2 induction being stronger in cancerous SCC than in normal CK cells. Moreover, SCC cells produced significantly more PGE(2) than CK cells, under both baseline and stimulated conditions (P < 0.05). NS-398, a selective COX-2 inhibitor, inhibited prostaglandin (PG)E(2) synthesis and decreased proliferation of CK and SCC cells (P < 0.05). Collectively, our results indicate that the canine neoplastic keratinocyte SCC cell line expresses more COX-2 and produces more PGE(2) than the normal keratinocyte CK cell line, thus providing an in vitro system to study the molecular basis of elevated COX-2 expression in SCCs in dogs.