Methotrexate Toxicity in Growing Long Bones of Young Rats: A Model for Studying Cancer Chemotherapy-Induced Bone Growth Defects in Children

The advancement and intensive use of chemotherapy in treating childhood cancers has led to a growing population of young cancer survivors who face increased bone health risks. However, the underlying mechanisms for chemotherapy-induced skeletal defects remain largely unclear. Methotrexate (MTX), the most commonly used antimetabolite in paediatric cancer treatment, is known to cause bone growth defects in children undergoing chemotherapy. Animal studies not only have confirmed the clinical observations but also have increased our understanding of the mechanisms underlying chemotherapy-induced skeletal damage. These models revealed that high-dose MTX can cause growth plate dysfunction, damage osteoprogenitor cells, suppress bone formation, and increase bone resorption and marrow adipogenesis, resulting in overall bone loss. While recent rat studies have shown that antidote folinic acid can reduce MTX damage in the growth plate and bone, future studies should investigate potential adjuvant treatments to reduce chemotherapy-induced skeletal toxicities

Prevention of methotrexate chemotherapy-induced bone growth arrest and osteoporosis with folinic acid.

During childhood and adolescence, bone continues to lengthen through endochondral ossification, which occurs within the growth plate and the adjacent metaphysis. As the production of calcified cartilage scaffold for bone deposition relies on the regulation of growth plate chondrocyte activities, any disruption to this carefully controlled process will result in bone growth defects. Methotrexate (MTX), an inhibitor of dihydrofolate reductase and DNA synthesis, is a commonly used chemotherapeutic agent in childhood oncology, and has been shown to induce bone growth defects in paediatric cancer patients and in short-term experimental young rats. Moreover, current knowledge on substances available to preserve bone growth during chemotherapy of childhood malignancies is limited. Previous animal studies have shown the short-term damaging effects of MTX on bone, and revealed that short-term MTX treatment in young rats can cause growth plate structural damages via suppression of chondrocyte proliferation and induction of chondrocyte apoptosis, which lead to metaphyseal bone loss. However, the underlying mechanisms for the structural and cellular damages remain unknown, particularly in the chronic treatment setting. Therefore, this PhD study, using chronic rat chemotherapy models, firstly aimed to compare and examine the damaging effects of low-dose vs. high-dose MTX on the skeleton and marrow progenitor cells of young rats. This was followed by mechanistic studies using immunostaining and real time RT-PCR with specimens from a chronic high-dose MTX chemotherapy trial, to identify underlying cellular and molecular mechanisms for MTX-induced growth plate and metaphyseal damages. In addition, this study also focused on the potential protective effects of supplementary anti-dote folinic acid (FA) against chronic MTX-induced skeletal damages. This study revealed chronic low-dose MTX treatment resulted in no damaging effects in the growth plate and nor significant suppression in primary spongiosa heights at the metaphysis. However, both short-term and chronic high-dose MTX treatment caused severe growth plate and metaphyseal damages. These results suggest MTXinduced skeletal toxicity in growing long bones is dose-dependent. Mechanistic studies using a chronic high-dose MTX chemotherapy model revealed that chronic MTX chemotherapy can result in severe structural and cellular damages at the growth plate. MTX was able to induce chondrocyte apoptosis, which was confirmed by real time RT-PCR analysis showing up-regulation of the apoptotic molecules. In addition, more cartilage resorptive cells “chondroclasts” were found along the cartilage-bone transitional zone after MTX treatment, which could affect the conversion of growth plate cartilage template into bone. In the metaphysis, MTX significantly reduced bone volume by inducing osteoblast apoptosis, adipocyte and osteoclast formation. However, molecular analysis within bone samples revealed no significant changes for molecules involved in bone cell differentiation, suggesting possible recovery of progenitors/ precursors after intense induction phase. However, some cytokines were found upregulated in blood plasma of treated rats. Finally, supplementary treatment with FA was able to reverse MTX-induced cellular damages at both the growth plate and metaphysis, suggesting FA supplementary treatment may be promising for reducing bone toxicity in young patients during chronic MTX chemotherapy.Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 201

Combination chemotherapy with cyclophosphamide, epirubicin and 5-fluorouracil causes trabecular bone loss, bone marrow cell depletion and marrow adiposity in female rats

The introduction of anthracyclines to adjuvant chemotherapy has increased survival rates among breast cancer patients. Cyclophosphamide, epirubicin and 5-fluorouracil (CEF) combination therapy is now one of the preferred regimens for treating node-positive breast cancer due to better survival with less toxicity involved. Despite the increasing use of CEF, its potential in causing adverse skeletal effects remains unclear. Using a mature female rat model mimicking the clinical setting, this study examined the effects of CEF treatment on bone and bone marrow in long bones. Following six cycles of CEF treatment (weekly intravenous injections of cyclophosphamide at 10 mg/kg, epirubicin at 2.5 mg/kg and 5-flurouracil at 10 mg/kg), a significant reduction in trabecular bone volume was observed at the metaphysis, which was associated with a reduced serum level of bone formation marker alkaline phosphatase (ALP), increased trends of osteoclast density and osteoclast area at the metaphysis, as well as an increased size of osteoclasts being formed from the bone marrow cells ex vivo. Moreover, a severe reduction of bone marrow cellularity was observed following CEF treatment, which was accompanied by an increase in marrow adipose tissue volume. This increase in marrow adiposity was associated with an expansion in adipocyte size but not in marrow adipocyte density. Overall, this study indicates that six cycles of CEF chemotherapy may induce some bone loss and severe bone marrow damage. Mechanisms for CEF-induced bone/bone marrow pathologies and potential preventive strategies warrant further investigation

Combination breast cancer chemotherapy with doxorubicin and cyclophosphamide damages bone and bone marrow in a female rat model

Published online: 26 May 2017Purpose: Anthracyclines (including doxorubicin) are still the backbone of commonly used breast cancer chemotherapy regimens. Despite increasing use of doxorubicin and cyclophosphamide (AC) combinations for treating breast cancer, their potential to cause adverse skeletal effects remains unclear. Methods: This study examined the effects of treatments with the AC regimen on bone and bone marrow in adult female rats. Results: AC treatment for four cycles (weekly intravenous injection of 2 mg/kg doxorubicin and 20 mg/kg cyclophosphamide) resulted in a reduced volume of trabecular bone at the metaphysis, which was associated with reduced serum levels of 25-hydroxy vitamin D3 and alkaline phosphatase. Reductions in densities of osteocytes and bone lining cells were also observed. In addition, bone marrow was severely damaged, including a severe reduction in bone marrow cellularity and an increase in marrow adipocyte content. Accompanying these changes, there were increases in mRNA expression of adipogenesis regulatory genes (PPARc and FABP4) and an inflammatory cytokine (TNFa) in metaphysis bone and bone marrow. Conclusions: This study indicates that AC chemotherapy may induce some bone loss, due to reduced bone formation, and bone marrow damage, due to increased marrow adiposity. Preventive strategies for preserving the bone and bone marrow microenvironment during anthracycline chemotherapy warrant further investigation.Chiaming Fan, Kristen R. Georgiou, Howard A. Morris, Ross A. McKinnon, Dorothy M. K. Keefe, Peter R. Howe, Cory J. Xia