Role of New Tyrosine Kinase Inhibitors in Osteoblastogenesis

Abstract

It has been reported that imatinib mesylate (IM) may affect bone tissue remodeling mainly by both an inhibitory activity on osteoclastogenesis and an induction of osteoblastogenesis. Dasatinib (DA) and Nilotinib (NI) are new generation tyrosine kinase inhibitors presently approved for chronic myeloid leukemia patients after imatinib failure. We therefore evaluated possible effects of DA and NI on osteoblatic differentiation of Mesenchymal Stem Cells derived from bone marrow (BM-MSCs). BM-MSCs are multipotent non-haematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, skeletal myocytes and nervous cells. Mesenchymal stem cells (hBM-MSCs) were obtained from bone marrow samples of normal healthy adult bone marrow donors, isolated by density gradient (mononuclear fraction) and cultured either in standard medium (SM) or in osteogenic medium (OM) (0.2 mM ascorbic acid, 0.1 μm dexamethasone and 10 mM β-glycerophosphate) with or without DA 2nM or NI 100nM. Osteogenic differentiation of hBM-MSCs was evaluated by changes in morphology, presence of mineralized nodules (evidenced by Alizarin red) and expression of osteoblast-associated genes such as osteocalcin (OCN), RUNX2 and Bone morphogenetic protein (BMP-2) evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by Scion Image. After 21days of culture, in comparison to control cultures, hBM-MSCs placed in OM, DA, NI and DA+OM, NI+OM exhibited changes in cell morphology from a spindle-shaped fibroblastic appearance to a rounder more cuboidal shape and the cells formed an extensive network of dense multilayered nodules (extracellular mineralization).Table I indicates mRNA expression of osteogenic markers in different culture conditions and shows that both DA and NI alone or in combination with OM, increase RUNX2, OCN, and BMP-2 expression.SMDANIOMDA + OMNI + OMRUNX21,59 0,202,09 0,164,2 0,312,86 0,254,41 0,414,18 0,24OCN2,57 0,283,2 0,143,14 0,093,59 0,173,6 0,283,62 0,25BMP-21,55 0,192,27 0,174,16 0,272,84 0,284,43 0,304,21 0,30SM= standard medium, OM= osteogenic medium, DA= dasatinib, NI= nilotinib In summary, our data show that both DA and NI, as already reported IM, may induce osteogenic differentiation of mesenchymal cells thus indicating that they potentially favour osteoblastogenesis.