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

Abstract

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.