Involvement of Cyclo-Oxygenase-2 in Osteoclast Formation and Bone Destruction in Bone Metastasis of Mammary Carcinoma Cell Lines

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Abstract
We previously reported that mouse mammary carcinoma cell lines (MMT060562 and BALB/c-MC) induced osteoclast formation through production of prostaglandin E2 (PGE2) in cocultures with mouse bone marrow cells, but the mechanism(s) of PG production remained unclear. In the present in vitro and in vivo studies, we tested the involvement of cyclo-oxygenase-2 (COX-2), an inducible rate-limiting enzyme in PG biosynthesis, in the stimulation of osteoclast formation by mouse mammary carcinoma cell lines. Addition of a selective COX-2 inhibitor, JTE-522, to cocultures of mammary carcinoma cell lines and bone marrow cells lowered PGE2 concentration in the culture media and inhibited osteoclast formation in a dose-dependent manner. Northern blotting showed a very high level of COX-2 messenger RNA (mRNA) expression in MMT060562. The mRNA expression was low in BALB/c-MC, but it increased when BALB/c-MC and bone marrow cells were cocultured. The results of immunocytochemistry for COX-2 protein in respective cultures were compatible with the results of COX-2 mRNA. In vivo, BALB/c-MC injected into the heart of Balb/c mice metastasized to bone and formed osteolytic lesions in their hindlimbs. Histological examination revealed that tumor cells had metastasized to the bone marrow cavity and destroyed the bone trabeculae. Immunohistochemistry demonstrated that bone marrow stromal cells adjacent to tumor cells expressed COX-2 protein. These findings suggest that COX-2 plays an important role in the osteolysis of bone metastasis in vivo as well as in osteoclast formation in cocultures used as an in vitro model of metastatic bone disease.