Inhibition of Osteosarcoma Cells Osteocalcin Counteracts Oxytocin-mediated Growth of A Plasmin-derived Hexapeptide from the Carboxyl End of Updated Version

We have previously described the presence of the functional plasminogen activator system on the surfaces of bone neoplastic cells and the fact that plasmin specifically cleaves bone matrix protein osteocalcin (OC). The cleavage of OC to NH2-midterminal (1–44) and COOH-terminal RFYGPV hexapeptide (44–49) proceeds with detachment of both products from bone mineral. Because the sequence of OC-derived hexapeptide (HP) is nearly identical to the E2 region of the oxytocin receptor (OTR), we set out to ascertain whether the HP interferes with the osteosarcoma (OS)-associated oxytocin (OT) system. We documented the presence and functional activity of OTRs in several OS cells by means of (a) OTmediated inhibition of OS growth; (b) expression of OTR mRNA by means of reverse transcription-PCR; (c) immunofluorescence staining with IF3 monoclonal antibody specific for human OTR; and (d) saturation binding and Scatchard analysis of OT binding to the receptors of isolated membranes or intact OS cells. Although we could not demonstrate direct binding of HP to OT, the presence of HP in cultures of OS cells antagonizes the inhibitory effect of OT on these cells. Additionally, in competitive binding assays, the HP effectively competes with binding of OT to its cognate receptors. The results indicate the existence of an OTR/OT system in tumor cells of bone origin. Suggested plasminogen activator-OCOTR/OT interactions may have an effect on the regulation of cell proliferation within the bone tissue as well as properties of the extracellular matrix surrounding the tumor foci in the bone.