Methotrexate treatment suppresses osteoblastic differentiation by inducing Notch2 signaling and blockade of Notch2 rescues osteogenesis by preserving Wnt/β‐catenin signaling

Abstract

Methotrexate (MTX) is a commonly used antimetabolite in cancer treatment. Its intensive use is linked with skeletal adverse effects such as reduced bone formation and bone loss, and yet little information is available on molecular mechanisms underlying MTX‐induced impaired bone formation. This study investigated the effects of MTX treatment at a clinical chemotherapy relevant dose on osteogenic differentiation in MC3T3E1 osteoblastic cells. To investigate the potential mechanisms, the expression of 87 genes regulating osteoblast differentiation and bone homeostasis was screened in MTX‐treated versus untreated cells by polymerase chain reaction (PCR) arrays and results illustrated significant upregulation of Notch2 and Notch target genes at both early and late stages of MC3T3E1 differentiation following MTX treatment. To confirm the roles of Notch2 pathway and its potential action mechanisms, MC3T3E1 cells were treated with MTX with an anti‐Notch2 neutralizing antibody or control IgG and effects were examined on osteogenesis and activation of the Wnt/β‐catenin pathway. Our results demonstrated that induction of Notch2 activity is associated with MTX adverse effects on osteogenic differentiation and blocking Notch2 rescues osteoblast differentiation by preserving activation of the Wnt/β‐catenin pathway.

Therapeutic Targeting Notch2 Protects Bone Micro-Vasculatures from Methotrexate Chemotherapy-Induced Adverse Effects in Rats

“…To examine the potential direct role of Notch2/Hey1 pathway in endothelial cell dysfunction, we conducted in vitro studies assessing treatment effects with MTX ± anti-Notch2 antibody in cultured rat bone marrow-derived endothelial cells (BMECs). Our results demonstrated that MTX exposure at a clinical chemotherapy relevant dose (10µM) [ 17 , 43 , 44 ] induced Notch2 activation with significantly increased mRNA expression levels of Notch ligand Jag1, Notch2 receptor, and Notch target gene Hey1 in cultured cells, and that supplementation of anti-Notch2 antibody suppressed MTX-induced Hey1 induction. Previously, over-expression of Hey1 was shown to inhibit proliferation, tube formation and migration in human endothelial cells [ 81 ].…”

“…To investigate the possible role of anti-Notch2 (NRR2) antibody in protecting endothelial cells against MTX damage, cells (at reaching 70% confluency) were treated with saline or MTX (10 µM) along with either the anti-ragweed antibody as control IgG (10 µg mL −1 ) or the anti-Notch2 antibody (10 µg mL −1 ) for 24 h, after which time the cells were harvested and conditioned medium was collected. The selected dose for MTX treatment in vitro is the chemotherapy relevant dose for MTX in cell culture studies [ 17 , 43 , 44 ]. The dose chosen for anti-Notch2 antibody treatment has previously been used in different in vitro studies to block Notch2 receptor activity effectively [ 36 , 44 , 45 ].…”

“…The selected dose for MTX treatment in vitro is the chemotherapy relevant dose for MTX in cell culture studies [ 17 , 43 , 44 ]. The dose chosen for anti-Notch2 antibody treatment has previously been used in different in vitro studies to block Notch2 receptor activity effectively [ 36 , 44 , 45 ].…”

Notch2 Blockade Mitigates Methotrexate Chemotherapy-Induced Bone Loss and Marrow Adiposity

“…Additionally, it has been illustrated that Notch2 induces osteoclastogenesis via enhancing expression of nuclear factor of activated T-cells 1 (NFATc1) [41]. Recently, in an in vitro study, we have found that MTX treatment-suppressed osteoblastic differentiation was associated with induced Notch2 signalling and Notch2 blockade attenuated MTX adverse effect in osteogenesis by activating Wnt/β-catenin signalling [42]. Despite previous studies on roles of Notch2 in skeletal development and diseases, there is a lack of knowledge about the potential roles of Notch2 signalling in cancer chemotherapy-induced bone defects.…”