Methotrexate Chemotherapy Promotes Osteoclast Formation in the Long Bone of Rats via Increased Pro-Inflammatory Cytokines and Enhanced NF-κB Activation

Cancer chemotherapy with methotrexate (MTX) is known to cause bone loss. However, the underlying mechanisms remain unclear. This study investigated the potential role of MIX-induced pro-inflammatory cytokines and activation of NF-kappa B in the associated osteoclastogenesis in rats. MTX (0.75 mg/kg per day) was administered for 5 days, and bone and bone marrow specimens were collected on days 6, 9, and 14. Compared with a normal control, MTX increased the density of osteoclasts within the metaphyseal bone and the osteoclast formation potential of marrow cells on day 9. RT-PCR analysis of mRNA expression for pro-osteoclastogenic cytokines in the metaphysis Indicated that, although the receptor activator of NF-kappa B ligand/osteoprotegerin axis was unaffected, expression of tumor necrosis factor (TNF)-alpha, IL-1, and IL-6 increased on day 9. Enzyme-linked immunosorbent assay analysis of plasma showed increased levels of TNF-alpha on day 6 and of IL-6 on day 14. Plasma from treated rats induced osteoclast formation from normal bone marrow cells, which was attenuated by a TNF-alpha-neutralizing antibody. Indicative of a role for NF-kappa B signaling, plasma on day 6 increased NF-kappa B activation in RAW(264.7) cells, and plasma-induced osteoclastogenesis was abolished in the presence of the NF-kappa B Inhibitor, parthenolide. Our results demonstrate mechanisms for MTX-induced osteoclastogenesis and show that MTX induces osteoclast differentiation by generating a pro-osteoclastogenic environment in both bone and the circulation, specifically with increased TNF-alpha levels and activation of NF-kappa B. (Am J Pathol 2014 181: 121-129; http://dx.doi.org/10.1016/j.ajpath.2012.03.037)