Among differentiating agents, purine bases and their corresponding nucleosides and nucleotides have been the center of focuses due to their important and diverse effects on many biological processes and also their physiological roles as extracellular signal molecules. Recent studies have proved that among purines, guanosine-base purines inhibit the excessive proliferation of a wide variety of cancer cells and guide them toward the maturation pathways. Based on these facts, anti-proliferative and osteogenic differentiation potential of guanosine 5ˈ-triphosphate (GTP) on human osteosarcoma cell line Saos-2 was evaluated. Cell viability was measured by tetrazolium-based colorimetric assay. Cell cycle was analyzed by propidium iodide staining using flow cytometry. Osteogenic differentiation responses were registered by assessment of alkaline phosphatase activity and the level of extracellular matrix mineralization which is detected and quantified by alizarin red S staining. Single dose treatment with variable concentrations of GTP revealed that GTP inhibits cell growth after 24 h of exposure and continued to increase in a time dependent manner, reaching a maximum value at 72-96 h of treatment. Cell cycle analyses by flow cytometry disclosed that growth inhibition was accompanied with an accumulation of cells in the S-phase of the cell cycle. Moreover, precise assessment showed that GTP has ability to induce osteogenic differentiation which was quantified by monitoring ALP activity and calcium deposition. A significant increase was observed in ALP activity in GTP (400 µM) treated cells as compared with control. Osteogenic differentiation was further confirmed by calcium mineralization under the influence of GTP. These finding may pave the way for further pharmaceutical evaluation of GTP as a suitable differentiating agent for poorly- differentiated osteosarcoma cells.

Keywords