Induzione del differenziamento osteoblastico in cellule di osteosarcoma umano MG63 mediante acido ascorbico

Ascorbic acid (AsA) is an important co-factor for the hydroxylation of proline and lysine residues in collagen and it is necessary for the differentiation of many cellular types, included osteoblasts. MG-63 is an osteoblast-like osteosarcoma lineage (OSC) often used for the study of osteoblast differentiation, but it is an atypical model since a low expression of some osteoblast specific transcription factors (Runx-2, Bone morphogenetic protein-Bmp-2) and proteins (bone alkaline phosphatase-BALP, osteocalcin-OC, type I collagen-Col1A1) and a low calcium deposition capability are observed. Some studies indicates that AsA increases BALP activity in OSC but its role in the differentiation process is not completely clear. The aim of our work is to study the effect of AsA in MG-63. Cells were treated with different AsA concentration (0,63, 125, 250 mcM) for 4 days. Then, we performed a proliferation assay (XTT), gene expression (RT-PCR) and protein (immunoblotting) analysis and immunocytochemistry evaluation. Alizaryn red assay for calcium deposition was performed after 6 days, and BALP colorimetric assessment every 2 days for 10 days. Our results showed a slight reduction of cell proliferation. At the same time, we observed an increased gene expression of Runx-2 and Osteopontin, no variation of Osteonectin, a reduction of Col1A1. These results were confirmed by the increased percentage of positive cells for Runx-2, Bmp2 and OC in immunocytochemistry, and by the increased Bmp-2 expression in immunoblotting analysis. The number of BALP positive cells increased progressively according with the duration of the treatment and Alizaryn red assay confirmed calcium deposition after therapy. These data, for the first time, show that AsA increases early osteoblastic specific transcription factors in OSC and they contribute to clarify a possible mechanism used by AsA to induce osteoblast differentiation. They give us also important information about OSC, in particular they confirm the crucial role of some transcription factors in tumorogenesis and in differentiation process. Finally, we could suggest that MG-63 are more osteoblast-like then original lineage after AsA treatment.