The 5-lipoxygenase inhibitor tepoxalin induces oxidative damage and altered PTEN status prior to apoptosis in canine osteosarcoma cell lines.

The 5-lipoxygenase (5-LOX) inhibitor tepoxalin has been shown to slow canine osteosarcoma (OSA) tumour xenografts growth, yet the mechanisms are poorly elucidated. Further examination of tepoxalin in canine OSA cell lines shows that tepoxalin treated cells undergo apoptosis through caspase-3 activation and annexin staining. Interestingly, apoptosis is superseded by an increase in reactive oxygen species (ROS), as measured by activation of dihydrorhodamine 123 and mitosox. This increase in ROS appears to be related to the 5-LOX inhibitor regardless of cellular 5-LOX status, and was not observed after treatment with the tepoxalin metabolite RWJ20142. Additionally, 5-LOX inhibition by tepoxalin appears to increase phosphatase and tensin (PTEN) homolog activity by preventing its alkylation or oxidation. PTEN modification or inhibition allows phosphoinositide-3 (PI3) kinase activity thereby heightening activation of protein kinase B (AKT) phosphorylation. Our data suggest that off target oxidation and LOX inhibition play roles in the apoptotic response.

Prevalence of 5-lipoxygenase expression in canine osteosarcoma and the effects of a dual 5-lipoxygenase/cyclooxygenase inhibitor on osteosarcoma cells in vitro and in vivo.

Canine osteosarcoma is an insidious disease with few effective treatment modalities; therefore, use of pharmacologic intervention to improve mortality or morbidity is constantly sought. The use of cyclooxygenase enzyme inhibitors has been an area of interest with limited efficacy based on retrospective examination of tumor expression and in vivo cell proliferation models. Recently, examination of dual cyclooxygenase and 5-lipoxygenase inhibitors in human and canine oncology suggests that 5-lipoxygenase inhibitors may be an effective approach in vitro and during tumor induction in rodent models. Therefore, the authors decided to examine 5-lipoxygenase expression in primary canine osteosarcoma samples and have shown that approximately 65% of osteosarcomas label positive for cytoplasmic 5-lipoxygenase. Further examination of a cell culture and xenograft model shows similar 5-lipoxygenase expression. Surprisingly, a canine 5-lipoxygenase inhibitor (tepoxalin) significantly reduced cell proliferation at physiologic doses in vitro and diminished xenograft tumor growth in nude mice, suggesting that further investigation is needed. Traditionally, 5-lipoxygense leads to production of lipid mediators, such as leukotriene B(4) and 5-oxo-eicosatetraenoic acid, which, when added back to the media of tepoxalin-treated cells, did not recover cell proliferation. The lack of nuclear staining in primary and xenografted tumors and the lack of response to eicoasanoids suggest that lipid mediator production is not the primary means by which tepoxalin acts to alter proliferation. Regardless of the mechanisms involved in retarding cell proliferation, future investigation is warranted.