Childhood methotrexate (MTX) chemotherapy often causes skeletal complications such as osteopenia and bone vasculature dysfunction. However, the precise molecular mechanism of damage is not completely understood and there is a lack of appropriate treatment. Notch signalling is one of the key signalling pathways known to regulate cell fate decision, proliferation and differentiation. Knowing the fact that bone vasculature plays crucial roles in bone turnover and bone homeostasis, whether and how Notch signalling pathway deregulation plays a role in cancer chemotherapy-induced bone damage and its correlation with vasculature disfunction is unknown.
Time course analyses of long bones from rats receiving intensive MTX treatment (5 consecutive daily doses of 0.75 mg/kg, which mimics childhood acute lymphoblastic leukemia treatment) found decreased trabecular bone volume, vasculature dilation and regression at day 9 following the first MTX dose. For exploring potential mechanisms, PCR array screening of the gene expression for 92 key factors regulating bone and vasculature homeostasis revealed increased expression of NOTCH2 after MTX treatment. Consistently, increased activity of Notch2 was confirmed by increased Notch2 intracellular domain protein level and by upregulation of Notch target genes in metaphysis at days 6 and 9 following the first MTX administration. To confirm the roles of Notch2 signalling in MTX bone and vasculature damage, a neutralising anti-Notch2 antibody or a control IgG was administrated during MTX treatment. Micro-CT analyses demonstrated that trabecular bone volume was preserved by the MTX+anti-Notch2 antibody treatment when compared to the MTX+Control IgG. Also, there was a significant increase in the volume of vasculature canals in the MTX+Control IgG treatment group compared to the control. However, blockade of Notch2+MTX treatment attenuated this increase. Our results suggest that Notch2 signalling plays an important role in mediating MTX treatment-induced bone damage/vasculature dysfunction and targeting Notch2 could be a potential treatment against MTX skeletal side effects.