Journal of Bone and Mineral Metabolism

, Volume 34, Issue 3, pp 277–290 | Cite as

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

  • Chiaming Fan
  • Kristen R. Georgiou
  • Ross A. McKinnon
  • Dorothy M. K. Keefe
  • Peter R. C. Howe
  • Cory J. Xian
Original Article


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.


Breast cancer chemotherapy Bone loss Marrow cellularity Marrow adiposity Osteoclast 



This project was funded in parts by the National Health Medical Research Council (NHMRC) of Australia and the University of South Australia. CJ Xian is a senior research fellow of NHMRC Australia. The authors thank Dr. Alice Lee for the advice on serum ALP assay.

Conflict of interest

The authors declare that there are no conflicts of interest.


  1. 1.
    Ackland SP, Anton A, Breitbach GP, Colajori E, Tursi JM, Delfino C, Efremidis A, Ezzat A, Fittipaldo A, Kolaric K, Lopez M, Viaro D, Group. Hs (2001) Dose-intensive epirubicin-based chemotherapy is superior to an intensive intravenous cyclophosphamide, methotrexate, and fluorouracil regimen in metastatic breast cancer: a randomized multinational study. J Clin Oncol 19:943–953PubMedGoogle Scholar
  2. 2.
    Gluck S (2005) Adjuvant chemotherapy for early breast cancer: optimal use of epirubicin. Oncologist 10:780–791CrossRefPubMedGoogle Scholar
  3. 3.
    Martin M, Villar A, Sole-Calvo A, Gonzalez R, Massuti B, Lizon J, Camps C, Carrato A, Casado A, Candel MT, Albanell J, Aranda J, Munarriz B, Campbell J, Diaz-Rubio E (2003) Doxorubicin in combination with fluorouracil and cyclophosphamide (i.v. FAC regimen, day 1, 21) versus methotrexate in combination with fluorouracil and cyclophosphamide (i.v. CMF regimen, day 1, 21) as adjuvant chemotherapy for operable breast cancer: a study by the GEICAM group. Ann Oncol 14:833–842CrossRefPubMedGoogle Scholar
  4. 4.
    Tack DK, Palmieri FM, Perez EA (2004) Anthracycline vs nonanthracycline adjuvant therapy for breast cancer. Oncology Williston Park 18:1367–1376PubMedGoogle Scholar
  5. 5.
    Laing K (2008) Managing the toxicities of the FEC-100 followed by docetaxel regimen: the price of success. Curr Oncol 15:112–113CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Jonat W, Kaufmann M, Sauerbrei W, Blamey R, Cuzick R, Namer M, Fogelman I, de Haes JC, de Matteis A, Stewart A, Eiermann W, Szakolczai I, Palmer M, Schumacher M, Geberth M, Lisboa B, Study. ZEBCRA (2002) Goserelin versus cyclophosphamide, methotrexate, and fluorouracil as adjuvant therapy in premenopausal patients with node-positive breast cancer: the Zoladex Early Breast Cancer Research Association Study. J Clin Oncol 20:4628–4635CrossRefPubMedGoogle Scholar
  7. 7.
    Shapiro CL, Manola J, Leboff M (2001) Ovarian failure after adjuvant chemotherapy is associated with rapid bone loss in women with early stage breast cancer. J Clin Oncol 19:3306–3311PubMedGoogle Scholar
  8. 8.
    Nurgalieva Z, Liu C-C, Du XL (2011) Chemotherapy use and risk of bone marrow suppression in a large population-based cohort of older women with breast and ovarian cancer. Med Oncol 28:716–725CrossRefPubMedGoogle Scholar
  9. 9.
    Georgiou KR, Hui SK, Xian CJ (2012) Regulatory pathways associated with bone loss and bone marrow adiposity caused by aging, chemotherapy, glucocorticoid therapy and radiotherapy. Am J Stem Cells 1:205–224PubMedPubMedCentralGoogle Scholar
  10. 10.
    Buttiglieri S, Ruella M, Risso A, Spatola T, Silengo L, Avvedimento EV, Tarella C (2011) The aging effect of chemotherapy on cultured human mesenchymal stem cells. Exp Hematol 39:1171–1181CrossRefPubMedGoogle Scholar
  11. 11.
    King TJ, Georgiou KR, Cool JC, Scherer MA, Ang ES, Foster BK, Xu J, Xian CJ (2012) Methotrexate chemotherapy promotes osteoclast formation in the long bone of rats via increased pro-inflammatory cytokines and enhanced NF-kappaB activation. Am J Pathol 181:121–129CrossRefPubMedGoogle Scholar
  12. 12.
    Raghu Nadhanan R, Abimosleh SM, Su YW, Scherer MA, Howarth GS, Xian CJ (2012) Dietary emu oil supplementation suppresses 5-fluorouracil chemotherapy-induced inflammation, osteoclast formation, and bone loss. Am J Physiol Endocrinol Metab 302:E1440–E1449CrossRefPubMedGoogle Scholar
  13. 13.
    Muralikrishnan G, Amalan Stanley V, Sadasivan Pillai K (2001) Dual role of vitamin C on lipid profile and combined application of cyclophosphamide, methotrexate and 5-fluorouracil treatment in fibrosarcoma-bearing rats. Cancer Lett 169:115–120CrossRefPubMedGoogle Scholar
  14. 14.
    Subramaniam S, Shyamala Devi CS (1995) Vitamin E protects intestinal basolateral membrane from CMF-induced damages in rat. Indian J Physiol Pharmacol 39:263–266PubMedGoogle Scholar
  15. 15.
    Burnell M, Levine MN, Chapman JA, Bramwell V, Gelmon K, Walley B, Vandenberg T, Chalchal H, Albain KS, Perez EA, Rugo H, Pritchard K, O’Brien P, Shepherd LE (2010) Cyclophosphamide, epirubicin, and Fluorouracil versus dose-dense epirubicin and cyclophosphamide followed by Paclitaxel versus Doxorubicin and cyclophosphamide followed by Paclitaxel in node-positive or high-risk node-negative breast cancer. J Clin Oncol 28:77–82CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Lee AM, Shandala T, Nguyen L, Muhlhausler BS, Chen KM, Howe PR, Xian CJ (2014) Effects of resveratrol supplementation on bone growth in young rats and microarchitecture and remodeling in ageing rats. Nutrients 6:5871–5887CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Fan C, Cool JC, Scherer MA, Foster BK, Shandala T, Tapp H, Xian CJ (2009) Damaging effects of chronic low-dose methotrexate usage on primary bone formation in young rats and potential protective effects of folinic acid supplementary treatment. Bone 44:61–70CrossRefPubMedGoogle Scholar
  18. 18.
    Hui SK, Sharkey L, Kidder LS, Zhang Y, Fairchild G, Coghill K, Xian CJ, Yee D (2012) The influence of therapeutic radiation on the patterns of bone marrow in ovary-intact and ovariectomized mice. PLoS One 7:e42668CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Albers J, Keller J, Baranowsky A, Beil FT, Catala-Lehnen P, Schulze J, Amling M, Schinke T (2013) Canonical Wnt signaling inhibits osteoclastogenesis independent of osteoprotegerin. J Cell Biol 200:537–549CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Fan CM, Foster BK, Hui SK, Xian CJ (2012) Prevention of bone growth defects, increased bone resorption and marrow adiposity with folinic acid in rats receiving long-term methotrexate. PLoS One 7:e46915CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Georgiou KR, King TJ, Scherer MA, Zhou H, Foster BK, Xian CJ (2012) Attenuated Wnt/beta-catenin signalling mediates methotrexate chemotherapy-induced bone loss and marrow adiposity in rats. Bone 50:1223–1233CrossRefPubMedGoogle Scholar
  22. 22.
    Raghu Nadhanan R, Skinner J, Chung R, Su YW, Howe PR, Xian CJ (2013) Supplementation with fish oil and genistein, individually or in combination, protects bone against the adverse effects of methotrexate chemotherapy in rats. PLoS One 8:e71592CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Murthy V, Chamberlain RS (2012) Menopausal symptoms in young survivors of breast cancer: a growing problem without an ideal solution. Cancer Control 19:317–329PubMedGoogle Scholar
  24. 24.
    Pacifici R (2008) Estrogen deficiency, T cells and bone loss. Cell Immunol 252:68–80CrossRefPubMedGoogle Scholar
  25. 25.
    Shapiro CL (2010) Estrogen deficiency and bone loss in women with breast cancer. Breast Cancer Res Treat 123:815–818CrossRefPubMedGoogle Scholar
  26. 26.
    Weitzmann MN, Pacifici R (2006) Estrogen deficiency and bone loss: an inflammatory tale. J Clin Invest 116:1186–1194CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Erselcan T, Kairemo KJ, Wiklund TA, Hernberg M, Blomqvist CP, Tenhunen M, Bergh J, Joensuu H (2000) Subclinical cardiotoxicity following adjuvant dose-escalated FEC, high-dose chemotherapy, or CMF in breast cancer. Br J Cancer 82:777–781CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Jenkins P, Wallis R (2010) Subclinical cardiotoxicity following adjuvant dose-escalated FEC, high-dose chemotherapy, or CMF in breast cancerDose-rounding of adjuvant chemotherapy for breast cancer: an audit of toxicity. J Oncol Pharm Practice 16:251–255CrossRefGoogle Scholar
  29. 29.
    Farquhar C, Marjoribanks J, Basser R, Hetrick S, Lethaby A (2005) High dose chemotherapy and autologous bone marrow or stem cell transplantation versus conventional chemotherapy for women with metastatic breast cancer. Cochrane Database Syst Rev, CD003142Google Scholar
  30. 30.
    von Wangenheim KH, Peterson HP, Cronkite EP, Feinendegen LE (1987) 5-Fluorouracil treatment after irradiation impairs recovery of bone marrow functions. Radiat Environ Biophys 26:163–170CrossRefGoogle Scholar
  31. 31.
    Ponnapakkam T, Katikaneni R, Nichols T, Tobin G, Sakon J, Matsushita O, Gensure RC (2011) Prevention of chemotherapy-induced osteoporosis by cyclophosphamide with a long-acting form of parathyroid hormone. J Endocrinol Invest 34:e392–e397PubMedGoogle Scholar
  32. 32.
    Sen AK, Karakas E, Bilaloglu R (2010) Genotoxic effect of epirubicin in mouse bone marrow in vivo. Z Naturforsch [C] 65:211–217CrossRefGoogle Scholar
  33. 33.
    Berliere M, Dalenc F, Malingret N, Vindevogel A, Piette P, Roche H, Donnez J, Symann M, Kerger J, Machiels JP (2008) Incidence of reversible amenorrhea in women with breast cancer undergoing adjuvant anthracycline-based chemotherapy with or without docetaxel. BMC Cancer 8:56CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Miller KK, Klibanski A (1999) Clinical review 106: amenorrheic bone loss. J Clin Endocrinol Metab 84:1775–1783PubMedGoogle Scholar
  35. 35.
    Xian CJ, Howarth GS, Cool JC, Foster BK (2004) Effects of acute 5-fluorouracil chemotherapy and insulin-like growth factor-I pretreatment on growth plate cartilage and metaphyseal bone in rats. Bone 35:739–749CrossRefPubMedGoogle Scholar
  36. 36.
    Bozec A, Bakiri L, Hoebertz A, Eferl R, Schilling AF, Komnenovic V, Scheuch H, Priemel M, Stewart CL, Amling M, Wagner EF (2008) Osteoclast size is controlled by Fra-2 through LIF/LIF-receptor signalling and hypoxia. Nature 454:221–225CrossRefPubMedGoogle Scholar
  37. 37.
    Fujita K, Iwasaki M, Ochi H, Fukuda T, Ma C, Miyamoto T, Takitani K, Negishi-Koga T, Sunamura S, Kodama T, Takayanagi H, Tamai H, Kato S, Arai H, Shinomiya K, Itoh H, Okawa A, Takeda S (2012) Vitamin E decreases bone mass by stimulating osteoclast fusion. Nat Med 18:589–594CrossRefPubMedGoogle Scholar
  38. 38.
    Lees RL, Sabharwal VK, Heersche JN (2001) Resorptive state and cell size influence intracellular pH regulation in rabbit osteoclasts cultured on collagen-hydroxyapatite films. Bone 28:187–194CrossRefPubMedGoogle Scholar
  39. 39.
    Mauch P, Constine L, Greenberger J, Knospe W, Sullivan J, Liesveld JL, Deeg HJ (1995) Hematopoietic stem cell compartment: acute and late effects of radiation therapy and chemotherapy. Int J Radiat Oncol Biol Phys 31:1319–1339CrossRefPubMedGoogle Scholar
  40. 40.
    Błogowski W, Ratajczak MZ, Zyżniewska-Banaszak E, Dołęgowska B, Starzyńska T (2012) Adipose tissue as a potential source of hematopoietic stem/progenitor cells. Obesity Silver Spring 20:923–931CrossRefPubMedGoogle Scholar
  41. 41.
    Naveiras O, Nardi V, Wenzel PL, Fahey F, Daley GQ (2009) Bone marrow adipocytes as negative regulators of the hematopoietic microenvironment. Nature 460:259–263CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Jones LC, Hungerford DS (2012) Bone marrow adipocytes: Friend or foe. J Bone Joint Surg Br 94B:60Google Scholar
  43. 43.
    Kitajima M, Shigematsu M, Ogawa K, Sugihara H, Hotokebuchi T (2007) Effects of glucocorticoid on adipocyte size in human bone marrow. Med Mol Morphol 40:150–156CrossRefPubMedGoogle Scholar
  44. 44.
    Miyanishi K, Yamamoto T, Irisa T, Yamashita A, Jingushi S, Noguchi Y, Iwamoto Y (2002) Bone marrow fat cell enlargement and a rise in intraosseous pressure in steroid-treated rabbits with osteonecrosis. Bone 30:185–190CrossRefPubMedGoogle Scholar
  45. 45.
    Motomura G, Yamamoto T, Miyanishi K, Yamashita A, Sueishi K, Iwamoto Y (2005) Bone marrow fat-cell enlargement in early steroid-induced osteonecrosis–a histomorphometric study of autopsy cases. Pathol Res Pract 200:807–811CrossRefPubMedGoogle Scholar
  46. 46.
    Shim K, MacKenzie MJ, Winquist E (2008) Chemotherapy-associated osteonecrosis in cancer patients with solid tumours: a systematic review. Drug Saf 31:359–371CrossRefPubMedGoogle Scholar

Copyright information

© The Japanese Society for Bone and Mineral Research and Springer Japan 2015

Authors and Affiliations

  • Chiaming Fan
    • 1
  • Kristen R. Georgiou
    • 1
  • Ross A. McKinnon
    • 1
    • 2
  • Dorothy M. K. Keefe
    • 1
    • 3
    • 4
  • Peter R. C. Howe
    • 5
  • Cory J. Xian
    • 1
  1. 1.School of Pharmacy and Medical Sciences, and Sansom Institute for Health ResearchUniversity of South AustraliaAdelaideAustralia
  2. 2.Flinders Centre for Innovation in Cancer, School of MedicineFlinders UniversityBedford ParkAustralia
  3. 3.SA Cancer ServiceSA Cancer Clinical Network, SA HealthAdelaideAustralia
  4. 4.Centre of Cancer MedicineUniversity of AdelaideAdelaideAustralia
  5. 5.Clinical Nutrition Research Centre, School of Biomedical Sciences and PharmacyUniversity of NewcastleCallaghanAustralia

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