Aims. Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. Methods. MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry. Results. Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and β1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells. Conclusion. Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion. Cite this article: Bone Joint Res 2024;13(4):157–168

Aim. In osteosarcoma, local control of the tumour is absolutely critical otherwise the chances of long term survival are <10% and may effectively approach zero. Radiotherapy is used in case of non-resectable tumours. Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation (XRT) by altering numerous molecular pathways. Therefore, we investigated the effect of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on radiation response in osteosarcoma (OS) and rhabdomyosarcoma (RMS) cell lines. Methods. Clonogenic survival, cell cycle analysis, apoptosis and gammaH2AX phosphorylation as a marker of DNA double strand breaks (DSBs) were examined in two OS (KHOS-24OS, SAOS2) and two RMS (A-204, RD) cell lines treated with SAHA alone and SAHA plus XRT, respectively. Protein expression was investigated via immunoblot analysis, cell cycle analysis, measurement of apoptosis and gammaH2AX expression were performed using flow cytometry. Results. In the sarcoma cell lines, SAHA induced an inhibition of cell proliferation and clonogenic survival and lead to a significant radiosensitization. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In both tumour entities, gammaH2AX expression was significantly increased when XRT was combined with SAHA treatment which was correlated with attenuation of radiation-induced DNA repair protein expression. Conclusion. Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair rather than increased apoptosis induction after exposure to HDACIs could be one key mechanism of radiosensitization by HDACIs

Aims

Bone metastasis ultimately occurs due to a complex multistep process, during which the interactions between cancer cells and bone microenvironment play important roles. Prior to colonization of the bone, cancer cells must succeed through a series of steps that will allow them to gain migratory and invasive properties; epithelial-to-mesenchymal transition (EMT) is known to be integral here. The aim of this study was to determine the effects of G protein subunit alpha Q (GNAQ) on the mechanisms underlying bone metastasis through EMT pathway.

The most appropriate protocol for the biopsy of musculoskeletal tumours is controversial, with some authors advocating CT-guided core biopsy. At our hospital the initial biopsies of most musculoskeletal tumours has been by operative core biopsy with evaluation by frozen section which determines whether diagnostic tissue has been obtained and, if possible, gives the definitive diagnosis. In order to determine the accuracy and cost-effectiveness of this protocol we have undertaken a retrospective audit of biopsies of musculoskeletal tumours performed over a period of two years.

A total of 104 patients had biopsies according to this regime. All gave the diagnosis apart from one minor error which did not alter the management of the patient. There was no requirement for re-biopsy. This protocol was more labour-intensive and 38% more costly than CT-guided core biopsy (AU$1804 vs AU$1308). However, the accuracy and avoidance of the anxiety associated with repeat biopsy outweighed these disadvantages.