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Bisphosphonates (BPs), such as pamidronate and clodronate, are an important class of drugs for the treatment of bone diseases. It is widely recognized that they inhibit bone resorption by suppressing the action of osteoclasts through antagonizing the mevalonate pathway, thereby reducing osteolytic bone metastases derived from different cancers, i.e. breast carcinoma and multiple myeloma. In contrast, the effects of BPs on primary bone tumors is an issue still to be resolved. Therefore, a systematic approach was set up to test the hypothesis that BPs could act directly on osteosarcoma cells. The effects of pamidronate and clodronate on seven osteosarcoma cell lines (HOS, MG-63, OST, SaOS-2, SJSA-1, U(2)OS and ZK-58) were studied. Pamidronate inhibited cell growth in a time- and dose-dependent manner, and decreased proliferation for up to 73% at 50 microM after 72 h, whereas its monophosphonate analog 3-aminopropyl phosphonate did not reduce cell viability at concentrations up to 2 mM. Clodronate showed less inhibitory effects (maximally 38% reduction at 1 mM after 72 h). Importantly, cell growth of fibroblasts was only very weakly affected by treatment with pamidronate. These results suggest that pamidronate may be a useful agent for the treatment of patients with osteosarcoma.

This study analyses the production of tumour necrosis factor (TNF)alpha and soluble TNF receptor (sTNF-R) before and after exposure to gamma irradiation and interferon gamma (IFN gamma) in 12 cell lines derived from Ewing's sarcoma (ES)/peripheral primitive neuroectodermal tumours (pPNET). Supernatants from ES/pPNET cell cultures were tested in a TNF alpha-specific amplified enzyme-linked immunosorbent assay (ELISA), a bioassay, and sTNF-Rp55 and sTNF-Rp75 ELISA. The tumour cell lines released minimal amounts of TNF alpha, prominent amounts of sTNF-Rp55 (7/12 cell lines) and no sTNF-Rp75. Exposure to gamma irradiation (5 Gy) either induced (3/12) cell lines) or up-regulated (3/12 cell lines) TNF alpha release without changing sTNF-Rp55 and sTNF-Rp75 levels. Priming of cultures with recombinant human IFN gamma (rhIFN gamma) markedly enhanced TNF alpha secretion in the radiation-responsive cell lines and had no influence on sTNF-Rp55 and sTNF-Rp75 levels. rhIFN gamma affected the magnitude rather than the sensitivity of the radiation response. The TNF alpha secreted was bioactive, as shown by its cytotoxic effect of WEHI-164 cells, and neutralization of its activity by anti-TNF alpha monoclonal antibody. Herbimycin A (a tyrosine-specific protein kinase inhibitor) but not calphostin C (a protein kinase C inhibitor), H89 (a protein kinase A inhibitor), AA-COCF3 (a specific inhibitor of phospholipase A2) and MK-886 (a specific inhibitor of 5-lipoxygenase) abrogated gamma-irradiation-stimulated TNF alpha release. The antioxidants N-acetylcysteine, nordihydroguaiaretic acid and mepacrine dose-dependently inhibited gamma-irradiation-mediated TNF alpha production. Collectively our findings indicate that IFN gamma priming potentiates the secretion of bioactive TNF alpha by ES/pPNET cells in response to gamma irradiation without affecting sTNF-R release. The data suggest a requirement for protein tyrosine kinase activity and a role for reactive oxygen species in the gamma-irradiation-mediated intracellular signalling pathway leading to TNF alpha production.