Antineoplastic effect of the cyclooxygenase inhibitor meloxicam on canine osteosarcoma cells

Abstract

A decisive role in cancer development has been attributed to cyclooxygenase-2 (COX-2) activity, but the significance of COX-2 inhibitors in cancer treatment still needs to be thoroughly investigated. We studied the influence of meloxicam, a non-steroidal antiinflammatory drug with preferential inhibitory effects on COX-2 compared to COX-1, on canine osteosarcoma (D-17) cells. We demonstrated that D-17 cells expressed mRNA and COX-2 protein. Treatment with meloxicam induced a time- and dose-dependent inhibition of cellular growth. To determine if apoptosis plays a role in meloxicam-induced cell death, we performed agarose gel electrophoresis and found a DNA-ladder pattern, typically seen in apoptosis, as well as early apoptotic changes by Annexin V tests. Furthermore, electron microscopy revealed ultrastructural alterations typical of apoptosis. Quantification of apoptotic cells by immunohistochemical staining of caspase 3 confirmed the results. However, further studies with meloxicam are necessary to assess its potential use for treatment of osteosarcomas in dogs.

Introduction

In dogs, osteosarcoma is the most frequently diagnosed primary bone tumor (Priester and Mc Kay, 1980). The prognosis of osteosarcoma after surgery is poor, whilst supplementary, expensive chemotherapeutics essentially prolong survival rates by controlling growth of distant metastases. However, their use is limited due to severe toxicity. Therefore, the ultimate goal remains to discover effective chemotherapeutics that are affordable and which have minimal toxicity.

Epidemiological studies recently provided evidence that cyclooxygenases might be involved in cancer development (Farrow et al., 1998). Until now, three isoforms of cyclooxygenases are known. Cyclooxygenase-1 (COX-1) was identified in many organs and cells, including as stomach, kidney, nervous system and platelets (O’Neill and Ford-Hutchinson, 1993). COX-1 is considered as a “housekeeping enzyme” that is involved in vascular haemostasis and gastroprotection. COX-3, an alternate splice variant of COX-1, is most abundant in the cerebral cortex and the heart (Chandrasekharan et al., 2002). COX-2 is expressed at very low levels in healthy tissues but its activity is increased markedly during inflammation (Vane et al., 1994). Moreover, increased COX-2 expression has been found in many human malignancies, such as colorectal (Eberhart et al., 1994), liver (Koga et al., 1999), pancreatic (Tucker et al., 1999), breast (Hwang et al., 1998), lung (Hida et al., 1998), bladder (Mohammed et al., 1999) primary tumors and metastases (Chen et al., 2001). COX-2 overexpression has been mentioned in connection with resistance to apoptosis (Tsujii and DuBois, 1995) and COX-2 activity is suspected to promote angiogenesis and tissue invasion of tumors (Tsujii et al., 1997, Tsujii et al., 1998).

Cyclooxygenases are responsible for the conversion of arachidonic acid to prostaglandins. Prostaglandins are of interest because of their possible role in oncogenesis (Fosslien, 2000). For example, prostanoid levels were increased during progression from adenoma to adenocarcinoma in patients with familial adenomatous polyposis (Yang et al., 1998). In dogs, prostaglandin E₂ levels were increased in osteosarcoma tissue samples compared to normal bone, and high concentrations of prostaglandin E₂ were found in the cell culture medium of cell lines derived from canine osteosarcoma lung metastases (Mohammed et al., 2001). Because of these findings, we investigated the antiproliferative effect of the cyclooxygenase inhibitor meloxicam, a drug licensed for veterinary use, on an established D-17 canine osteosarcoma cell line expressing COX-2. We measured viable cells via proliferation assays after 24, 48 and 72 h of meloxicam exposure. In addition, we wanted to define the nature of meloxicam-induced cell death more precisely.