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 MTX-induced pro-inflammatory cytokines and activation of NF-κB 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-κB ligand/osteoprotegerin axis was unaffected, expression of tumor necrosis factor (TNF)-α, IL-1, and IL-6 increased on day 9. Enzyme-linked immunosorbent assay analysis of plasma showed increased levels of TNF-α 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-α-neutralizing antibody. Indicative of a role for NF-κB signaling, plasma on day 6 increased NF-κB activation in RAW(264.7) cells, and plasma-induced osteoclastogenesis was abolished in the presence of the NF-κB 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-α levels and activation of NF-κB.

Methotrexate chemotherapy reduces osteogenesis but increases adipogenic potential in the bone marrow.

Intensive use of cancer chemotherapy is increasingly linked with long-term skeletal side effects such as osteopenia, osteoporosis and fractures. However, cellular mechanisms by which chemotherapy affects bone integrity remain unclear. Methotrexate (MTX), used commonly as an anti-metabolite, is known to cause bone defects. To study the pathophysiology of MTX-induced bone loss, we examined effects on bone and marrow fat volume, population size and differentiation potential of bone marrow stromal cells (BMSC) in adult rats following chemotherapy for a short-term (five once-daily doses at 0.75 mg/kg) or a 6-week term (5 doses at 0.65 mg/kg + 9 days rest + 1.3 mg/kg twice weekly for 4 weeks). Histological analyses revealed that both acute and chronic MTX treatments caused a significant decrease in metaphyseal trabecular bone volume and an increase in marrow adipose mass. In the acute model, proliferation of BMSCs significantly decreased on days 3-9, and consistently the stromal progenitor cell population as assessed by CFU-F formation was significantly reduced on day 9. Ex vivo differentiation assays showed that while the osteogenic potential of isolated BMSCs was significantly reduced, their adipogenic capacity was markedly increased on day 9. Consistently, RT-PCR gene expression analyses showed osteogenic transcription factors Runx2 and Osterix (Osx) to be decreased but adipogenic genes PPARγ and FABP4 up-regulated on days 6 and 9 in the stromal population. These findings indicate that MTX chemotherapy reduces the bone marrow stromal progenitor cell population and induces a switch in differentiation potential towards adipogenesis at the expense of osteogenesis, resulting in osteopenia and marrow adiposity.

Damaging effects of chronic low-dose methotrexate usage on primary bone formation in young rats and potential protective effects of folinic acid supplementary treatment.

Methotrexate (MTX) is a most commonly used anti-metabolite in cancer treatment and as an anti-rheumatic drug. While MTX chemotherapy at a high dose is known to cause bone growth defects in growing bones, effects of its chronic use at a low dose on growing skeleton remain less clear. Here, we examined effects on bone growth of long-term MTX chemotherapy at a low dose in young rats, and potential protective effects of supplementary treatment with antidote folinic acid (given ip at 1 mg/kg 6 h after MTX). After two cycles of 5 once-daily MTX injections (at 0.75 mg/kg, 5 days on/9 days off/5 days on), histological analysis showed that MTX at this dose caused significant reduction in heights of growth plate and primary spongiosa bone on day 22 compared to controls (P<0.05). In contrast, a similar dosing regimen but at a lower dose (0.4 mg/kg) caused only slight or no reduction in heights of both regions. However, after the induction phase at this 0.4 mg/kg dosing, continued use of MTX at a low dose (once weekly at 0.2 mg/kg) caused a reduction in primary spongiosa height and bone volume on weeks 9 and 14, which was associated with an increased osteoclast formation and their bone surface density as well as a decreased osteoblast bone surface density in the primary spongiosa. Folinic acid supplementation was shown able to prevent the MTX effects in the primary spongiosa. These results suggest that acute use of MTX can damage growth plate and primary bone at a high dose, but not at a low dose. However, long-term use of MTX at a low dose can reduce primary bone formation probably due to decreased osteoblastic function but increased osteoclastic formation and function, and supplementary treatment with folinic acid may be potentially useful in protecting bone growth during long-term low-dose MTX chemotherapy.

Folinic acid attenuates methotrexate chemotherapy-induced damages on bone growth mechanisms and pools of bone marrow stromal cells.

Chemotherapy often induces bone growth defects in pediatric cancer patients; yet the underlying cellular mechanisms remain unclear and currently no preventative treatments are available. Using an acute chemotherapy model in young rats with the commonly used antimetabolite methotrexate (MTX), this study investigated damaging effects of five once-daily MTX injections and potential protective effects of supplementary treatment with antidote folinic acid (FA) on cellular activities in the tibial growth plate, metaphysis, and bone marrow. MTX suppressed proliferation and induced apoptosis of chondrocytes, and reduced collagen-II expression and growth plate thickness. It reduced production of primary spongiosa bone, volume of secondary spongiosa bone, and proliferation of metaphyseal osteoblasts, preosteoblasts and bone marrow stromal cells, with the cellular activities being most severely damaged on day 9 and returning to or towards near normal levels by day 14. On the other hand, proliferation of marrow pericytes was increased early after MTX treatment and during repair. FA supplementation significantly suppressed chondrocyte apoptosis, preserved chondrocyte proliferation and expression of collagen-II, and attenuated damaging effects on production of calcified cartilage and primary bone. The supplementation also significantly reduced MTX effects on proliferation of metaphyseal osteoblastic cells and of bone marrow stromal cells, and enhanced pericyte proliferation. These observations suggest that FA supplementation effectively attenuates MTX damage on cellular activities in producing calcified cartilage and primary trabecular bone and on pools of osteoblastic cells and marrow stromal cells, and that it enhances proliferation of mesenchymal progenitor cells during bone/bone marrow recovery.

Cellular mechanisms for methotrexate chemotherapy-induced bone growth defects.

Methotrexate (MTX) is a commonly used anti-metabolite in childhood oncology and is known to cause bone growth arrest and osteoporosis; yet the underlying mechanisms for MTX-induced bone growth defects remain largely unclear. This study characterized damaging effects in young rats of acute chemotherapy with 5 once-daily doses of MTX (0.75 mg/kg) on the cellular activities in the growth plate in producing calcified cartilage and trabecular bone and on activities of osteoblastic cells in the metaphysis. MTX treatment significantly induced chondrocyte apoptosis. MTX also suppressed chondrocyte proliferation and reduced collagen-II mRNA expression and total thickness of the growth plate, with the damage being most obvious on day 9 after the first injection, and with the growth plate histological structure returning normal on day 14. In the adjacent metaphyseal bone, mirroring the decrease in the width of the growth plate, production of primary spongiosa bone was markedly reduced and bone volume of the secondary spongiosa was decreased. Furthermore, MTX treatment significantly induced osteocyte apoptosis in the primary spongiosa and reduced proliferation of osteoblasts and preosteoblasts particularly in the secondary spongiosa. These observations suggest that methotrexate chemotherapy may cause bone growth defects by arresting cellular activities in the growth plate in producing calcified cartilage and primary trabecular bone and by decreasing pools of metaphyseal osteoblastic cells. However, this short-term MTX treatment only caused transit suppressions on growth plate cartilage and trabecular bone, as most cellular and histological parameters had recovered by day 14 or 21.

Effects of etoposide and cyclophosphamide acute chemotherapy on growth plate and metaphyseal bone in rats.

Pediatric cancer chemotherapy is known to cause bone growth arrest and osteoporotic changes, and yet the underlying mechanisms remain largely unknown. This project investigated effects of acute chemotherapy with topoisomerase inhibitor etoposide (Eto, 80 mg/kg), alkylating agent cyclophosphamide (Cyc, 240 mg/kg) or their combination (Cyc 120 mg/kg + Eto 50 mg/kg) on structural and cellular changes in the growth plate cartilage and metaphyseal bone, two important regions responsible for bone growth and bone mass accumulation. On day 3 after a single injection with either of the three treatments, although the total growth plate thickness was not significantly altered, the cellularity and height of the proliferative zone were significantly reduced. It was shown that while Eto suppressed chondrocyte proliferation, Cyc induced apoptosis in the growth plate proliferative zone. In the metaphysis, although osteoblastic cell surface was decreased in all three treated groups, the trabecular bone bone volume (BV/TV%) was not significantly altered on day 3. On the other hand, the acute chemotherapy reduced heights of both primary and secondary spongiosa trabecular bone. Therefore, Eto and/ or Cyc chemotherapy altered survival or proliferation of growth plate chondrocytes and metaphyseal osteoblastic cells and reduced heights of metaphyseal spongiosa trabecular bone, which may contribute to chemotherapy side effects of these two drugs on bone lengthening and bone mass accumulation.

Restricted fetal growth and lung development: a morphometric analysis of pulmonary structure.

Intrauterine growth restriction (IUGR) in humans increases the risk of lung disease and impaired function suggesting that adverse intra-uterine conditions can alter lung development. We hypothesized that placental restriction (PR) of fetal growth would alter lung structure in late gestation. PR involved removal of implantation sites in pre-pregnant ewes. Normal (n = 7) and PR (n = 11) fetuses were delivered at day 140 gestation. Lungs were fixed by tracheal infusion, processed and analyzed by morphometry. PR reduced ponderal index (PI) of lambs by 13%, increased lung volume:body weight (BW) (19%), and decreased the proportion of lung volume that comprised parenchyma from 86.5(2.6)% to 76.7(2.1)% with no change in absolute volume of non-parenchyma. Within the parenchyma, PR increased the proportion comprising airspace from 42.0(2.2)% to 55.5(1.7)% with smaller (-13%) more dense (18%) airsacs/alveoli present. The overall effect was a reduction in total gas-exchange surface density (-10%). Lung wet-weight and volume, parenchymal volume, gas-exchange tissue, and airspace volumes and gas-exchange surface area correlated positively with BW and crown-rump length (CRL) for all animals. The relative lung weight and volume correlated negatively with BW, CRL, and lung weight:BW with PI. Lung weight, lung volume, parenchymal volume, airspace perimeter, percent of parenchymal gas-exchange tissue, gas-exchange surface density, and area correlated positively with PI. The results indicate increased sparing of lung growth but with increasing structural changes, predominantly within lung parenchyma, with increasing growth restriction. Structural alterations associated with PR and poor fetal growth may be important in the pathogenesis of impaired lung function associated with IUGR.

Roles of neutrophil-mediated inflammatory response in the bony repair of injured growth plate cartilage in young rats.

Injured growth plate cartilage is often repaired by bony tissue, resulting in impaired bone growth in children. Previously, injury-induced, initial inflammatory response was shown to be an acute inflammatory event containing predominantly neutrophils. To examine potential roles of neutrophils in the bony repair, a neutrophil-neutralizing antiserum or control normal serum was administered systemically in rats with growth plate injury. The inflammatory response was found temporally associated with increased expression of neutrophil chemotactic chemokine cytokine-induced neutrophil chemoattractant-1 and cytokines TNF-alpha and IL-1beta. Following the inflammatory response, mesenchymal infiltration, chondrogenic and osteogenic responses, and bony repair were observed at the injury site. Neutrophil reduction did not significantly affect infiltration of other inflammatory cells and expression of TNF-alpha and IL-1beta and growth factors, platelet-derived growth factor-B and TGF-beta1, at the injured growth plate on Day 1 and had no effects on mesenchymal infiltration on Day 4. By Day 10, however, there was a significant reduction in proportion of mesenchymal repair tissue but an increase (although statistically insignificant) in bony trabeculae and a decrease in cartilaginous tissue within the injury site. Consistently, in antiserum-treated rats, there was an increase in expression of osteoblastic differentiation transcription factor cbf-alpha1 and bone matrix protein osteocalcin and a decrease in chondrogenic transcription factor Sox-9 and cartilage matrix collagen-II in the injured growth plate. These results suggest that injury-induced, neutrophil-mediated inflammatory response appears to suppress mesenchymal cell osteoblastic differentiation but enhance chondrogenic differentiation, and thus, it may be involved in regulating downstream chondrogenic and osteogenic events for growth plate bony repair.

Damage and recovery of the bone growth mechanism in young rats following 5-fluorouracil acute chemotherapy.

Chemotherapy-induced bone growth arrest and osteoporosis are significant problems in paediatric cancer patients, and yet how chemotherapy affects bone growth remains unclear. This study characterised development and resolution of damage caused by acute chemotherapy with antimetabolite 5-fluorouracil (5-FU) in young rats in the growth plate cartilage and metaphyseal bone, two important tissues responsible for bone lengthening. In metaphysis, 5-FU induced apoptosis among osteoblasts and preosteoblasts on days 1-2. In growth plate, chondrocyte apoptosis appeared on days 5-10. Interestingly, Bax was induced prior to apoptosis and Bcl-2 was upregulated during recovery. 5-FU also suppressed cell proliferation on days 1-2. While proliferation returned to normal by day 3 in metaphysis, it recovered partially on day 3, overshot on days 5-7 and normalised by day 10 in growth plate. Histologically, growth plate heights decreased by days 4-5 and returned normal by day 10. In metaphysis, primary spongiosa height was also reduced, mirroring changes in growth plate thickness. In metaphyseal secondary spongiosa, a reduced bone volume was observed on days 7-10 as there were fewer but more separated trabeculae. Starting from day 4, expression of some cartilage/bone matrix proteins and growth factors (TGF-beta1 and IGF-I) was increased. By day 14, cellular activity, histological structure and gene expression had returned normal in both tissues. Therefore, 5-FU chemotherapy affects bone growth directly by inducing apoptosis and inhibiting proliferation at growth plate cartilage and metaphyseal bone; after the acute damage, bone growth mechanism can recover, which is associated with upregulated expression of matrix proteins and growth factors.

Pre-treatment with insulin-like growth factor-I partially ameliorates 5-fluorouracil-induced intestinal mucositis in rats.

Insulin-like growth factor-I (IGF-I) has been demonstrated to enhance mucosal repair following intestinal damage induced by chemotherapeutic agents (intestinal mucositis). However, the potential for prophylactic IGF-I to protect the intestine remains undefined. We investigated the effects of IGF-I pre-treatment on chemotherapy-induced mucositis in rats. Male Sprague Dawley rats were treated for 7 days with 0 or 4.3mg/kg/day IGF-I delivered systemically via osmotic mini-pump. Rats received an intraperitoneal injection of 0 or 150 mg/kg 5-fluorouracil (5-FU) on day 7 and were killed 48 h later for assessment of intestinal damage and repair. Compared to normal controls, 5-FU decreased epithelial proliferation by 86%, concurrently increasing the incidence of apoptosis 87-fold, whilst decreasing small intestinal (SI) length by 14%, SI weight by 30% and total gut weight by 24%. 5-FU decreased villus height in the duodenum (23%), jejunum (20%) and ileum (30%) with crypt depths decreased by 31%, 27% and 33% in these gut regions. These effects were less profound in IGF-I pre-treated rats in which apoptosis was increased 48-fold, with SI length decreased by 7%, SI weight by 18% and total gut weight by 15% accompanied by decreases in villus height of 8% (duodenum), 14% (jejunum) and 21% (ileum), and crypt depth decreases of 23%, 16% and 17% for the same gut regions, compared to normal controls. We conclude that IGF-I pre-treatment only partially attenuates features of intestinal mucositis when assessed 48 h after 5-FU chemotherapy.