Clinical and Experimental Medicine

, Volume 17, Issue 2, pp 151–160 | Cite as

Effects of lifelong exercise training on mammary tumorigenesis induced by MNU in female Sprague–Dawley rats

  • Ana I. Faustino-RochaEmail author
  • Adelina Gama
  • Paula A. Oliveira
  • Antonieta Alvarado
  • Maria J. Neuparth
  • Rita Ferreira
  • Mário Ginja
Original Article


Breast cancer is the most common malignancy in women worldwide. Several studies have suggested that exercise training may decrease the risk of breast cancer development. This study aimed to evaluate the effects of long-term exercise training on mammary tumorigenesis in an animal model of mammary cancer. Fifty female Sprague–Dawley rats were randomly divided into four groups: MNU sedentary, MNU exercised, control sedentary and control exercised. Animals from MNU groups received an intraperitoneal administration of N-methyl-N-nitrosourea (MNU). Animals were exercised on a treadmill during 35 weeks. When animals were killed, blood samples were collected to determine the hematocrit and to perform the biochemical analysis. Mammary tumors were collected and histologically evaluated; the expression of ERs α and β was evaluated in tumor sections by immunohistochemistry. All survived animals from both MNU groups developed mammary tumors. The number of mammary tumors (p > 0.05) and lesions (p = 0.056) was lower in MNU exercised than in MNU sedentary animals. MNU exercised animals showed lower number of malignant lesions than MNU sedentary animals (p = 0.020). C-reactive protein serum concentration was lower in exercised animals; however, the levels of 17-β estradiol were higher in exercised animals. Tumors from exercised animals exhibited higher expression of ER α than tumors from sedentary animals (p < 0.05). This study analyzes the impact of the longest exercise training protocol on mammary tumorigenesis ever performed. We concluded that the lifelong endurance training has beneficial effects on mammary tumorigenesis in female rats (reduced the inflammation, the number of mammary tumors and lesions, and histological grade of malignancy). Additionally, the mammary tumors from MNU exercised group exhibited higher immunoexpression of ER α that is an indicator of well-differentiated tumors and better response to hormone therapy.


Estradiol Estrogen receptors Exercise Mammary tumors MNU 





Estrogen receptor


Mortality index


Tumors’ volume




C-reactive protein


Hematoxylin and eosin


Standard deviation


High-density lipoprotein


Low-density lipoprotein


Alanine aminotransferase


Creatine kinase



This work was supported by European Investment Funds by FEDER/COMPETE/POCI—Operational Competitiveness and Internationalization Program, under project POCI-01-0145-FEDER-006958, and Portuguese Foundation for Science and Technology (FCT), under the project UID/AGR/04033/2013, the project PTDC/DES/114122/2009 and post-graduation grant SFRH/BD/102099/2014.

Authors’ contributions

All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.


  1. 1.
    Harvie M, Howell A, Evans D. Can diet and lifestyle prevent breast cancer: what is the evidence? ASCO. 2015;35:66–73.Google Scholar
  2. 2.
    Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer J Clin. 2012;62:10–29.CrossRefPubMedGoogle Scholar
  3. 3.
    Brekelmans CTM. Risk factors and risk reduction of breast and ovarian cancer. Curr Opin Obstet Gynecol. 2003;15:63–8.CrossRefPubMedGoogle Scholar
  4. 4.
    Hunter DJ, Kelsey KT. Pesticide-residues and breast-cancer: the Harvest of a silent spring. J Natl Cancer Inst. 1993;85:598–9.CrossRefPubMedGoogle Scholar
  5. 5.
    Kelsey JL, Gammon MD. Epidemiology of breast-cancer. Epidemiol Rev. 1990;12:228–40.CrossRefPubMedGoogle Scholar
  6. 6.
    MacMahon B. Epidemiology and the causes of breast cancer. Int J Cancer. 2006;118:2373–8.CrossRefPubMedGoogle Scholar
  7. 7.
    Payne AH, Hales DB. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev. 2004;25:947–70.CrossRefPubMedGoogle Scholar
  8. 8.
    Ghayee HK, Auchus RJ. Basic concepts and recent developments in human steroid hormone biosynthesis. Rev Endocr Metab Disord. 2007;8:289–300.CrossRefPubMedGoogle Scholar
  9. 9.
    Simpson ER. Sources of estrogen and their importance. J Steroid Biochem. 2003;86:225–30.CrossRefGoogle Scholar
  10. 10.
    Kuiper GGJM, Carlsson B, Grandien K, et al. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology. 1997;138:863–70.Google Scholar
  11. 11.
    Miyakoshi T, Miyajima K, Takekoshi S, Osamura RY. The influence of endocrine disrupting chemicals on the proliferation of ER alpha knockdown-human breast cancer cell line MCF-7; new attempts by RNAi technology. Acta Histochem Cytochem. 2009;42:23–8.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Saji S, Jensen EV, Nilsson S, Rylander T, Warner M, Gustafsson JA. Estrogen receptors alpha and beta in the rodent mammary gland. Proc Natl Acad Sci USA. 2000;97:337–42.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Rastelli F, Crispino S. Factors predictive of response to hormone therapy in breast cancer. Tumori. 2008;94:370–83.PubMedGoogle Scholar
  14. 14.
    Ma CX, Sanchez CG, Ellis MJ. Predicting endocrine therapy responsiveness in breast cancer. Oncology. 2009;23:133–42.PubMedGoogle Scholar
  15. 15.
    Morani A, Warner M, Gustafsson JA. Biological functions and clinical implications of oestrogen receptors alfa and beta in epithelial tissues. J Intern Med. 2008;264:128–42.CrossRefPubMedGoogle Scholar
  16. 16.
    Coyle Y. Physical activity as a negative modulator of estrogen-induced breast cancer. Cancer Causes Control. 2008;19:1021–9.CrossRefPubMedGoogle Scholar
  17. 17.
    Adams SA, Matthews CE, Hebert JR, et al. Association of physical activity with hormone receptor status: the Shanghai Breast Cancer Study. Cancer Epidemiol Biomark. 2006;15:1170–8.CrossRefGoogle Scholar
  18. 18.
    Jones LW, Eves ND, Courneya KS, Chiu BK, Baracos VE, Hanson J. Effects of exercise training on antitumor efficacy of doxorubicin in MDA-MB-231 breast cancer xenografts. Clin Cancer Res. 2005;11:6695–8.CrossRefPubMedGoogle Scholar
  19. 19.
    McTiernan A, Ulrich C, Slate S, Potter J. Physical activity and cancer etiology: associations and mechanisms. Cancer Cause Control. 1998;9:487–509.CrossRefGoogle Scholar
  20. 20.
    Tehard B, Friedenreich CM, Oppert JM, Clavel-Chapelon F. Effect of physical activity on women at increased risk of breast cancer: results from the E3 N cohort study. Cancer Epidemiol Biomark. 2006;15:57–64.CrossRefGoogle Scholar
  21. 21.
    Westerlind KC, McCarty HL, Gibson KJ, Strange R. Effect of exercise on the rat mammary gland: implications for carcinogenesis. Acta Physiol Scand. 2002;175:147–56.CrossRefPubMedGoogle Scholar
  22. 22.
    Westerlind KC, McCarty HL, Schultheiss PC, et al. Moderate exercise training slows mammary tumour growth in adolescent rats. Eur J Cancer Prev. 2003;12:281–7.CrossRefPubMedGoogle Scholar
  23. 23.
    Oberbach A, Lehmann S, Kirsch K, et al. Long-term exercise training decreases interleukin-6 (IL-6) serum levels in subjects with impaired glucose tolerance: effect of the-174G/C variant in IL-6 gene. Eur J Endocrinol. 2008;159:129–36.CrossRefPubMedGoogle Scholar
  24. 24.
    Forbes D, Blom H, Kostomitsopulos N, Moore G, Perretta G. Euroguide: on the accommodation and care of animals used for experimental and other scientific purposes. London: FELASA; 2007.Google Scholar
  25. 25.
    Faustino-Rocha A, Silva A, Gabriel J, et al. Ultrasonographic, thermographic and histologic evaluation of MNU-induced mammary tumors in female Sprague-Dawley rats. Biomed Pharmacother. 2013;67:771–6.CrossRefPubMedGoogle Scholar
  26. 26.
    Russo J, Russo IH. Atlas and histologic classification of tumors of the rat mammary gland. J Mammary Gland Biol. 2000;5:187–200.CrossRefGoogle Scholar
  27. 27.
    Ting AY, Kimler BF, Fabian CJ, Petroff BK. Characterization of a preclinical model of simultaneous breast and ovarian cancer progression. Carcinogenesis. 2007;28:130–5.CrossRefPubMedGoogle Scholar
  28. 28.
    Ueno T, Toi M, Saji H, et al. Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer. Clin Cancer Res. 2000;6:3282–9.PubMedGoogle Scholar
  29. 29.
    Murphy EA, Davis JM, Barrilleaux TL, McClellan JL, Steiner JL, Carmichael MD. Benefits of exercise training on breast cancer progression and inflammation in C3(1)SV40Tag mice. Cytokine. 2011;55:274–9.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Allred CD, Allred KF, Ju YH, et al. Dietary genistein results in larger MNU-induced, estrogen-dependent mammary tumors following ovariectomy of Sprague-Dawley rats. Carcinogenesis. 2004;25:211–8.CrossRefPubMedGoogle Scholar
  31. 31.
    Russo J, Russo IH. Experimentally induced mammary tumors in rats. Breast Cancer Res Treat. 1996;39:7–20.CrossRefPubMedGoogle Scholar
  32. 32.
    Whittal-Strange KS, Chadau S, Parkhouse WS. Exercise during puberty and NMU induced mammary tumorigenesis in rats. Breast Cancer Res Treat. 1998;47:1–8.CrossRefPubMedGoogle Scholar
  33. 33.
    Leygue E, Dotzlaw H, Watson PH, Murphy LC. Altered estrogen receptor alpha and beta messenger RNA expression during human breast tumorigenesis. Cancer Res. 1998;58:3197–201.PubMedGoogle Scholar
  34. 34.
    Roger P, Sahla ME, Makela S, Gustafsson JA, Baldet P, Rochefort H. Decreased expression of estrogen receptor beta protein in proliferative preinvasive mammary tumors. Cancer Res. 2001;61:2537–41.PubMedGoogle Scholar
  35. 35.
    Zeleniuch-Jacquotte A, Toniolo P, Levitz M, et al. Endogenous estrogens and risk of breast cancer by estrogen receptor status: a prospective study in postmenopausal women. Cancer Epidemiol Biomark. 1995;4:857–60.Google Scholar
  36. 36.
    Padrao AI, Moreira-Goncalves D, Oliveira PA, et al. Endurance training prevents TWEAK but not myostatin-mediated cardiac remodelling in cancer cachexia. Arch Biochem Biophys. 2015;567:13–21.CrossRefPubMedGoogle Scholar
  37. 37.
    Faustino-Rocha AI, Silva A, Gabriel J, Gil da Costa RM, Moutinho M, Oliveira PA,Gama A, Ferreira R, Ginja M. Long-term exercise training as a modulator of mammary cancer cascularization. Biomed Pharmacother. 2016;(Accepted).Google Scholar
  38. 38.
    Leung YK, Lee MT, Lam HM, Tarapore P, Ho SM. Estrogen receptor-beta and breast cancer: translating biology into clinical practice. Steroids. 2012;77:727–37.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Fox EM, Davis RJ, Shupnik MA. ER beta in breast cancer: onlooker, passive player, or active protector? Steroids. 2008;73:1039–51.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Murphy L, Cherlet T, Lewis A, Banu Y, Watson P. New insights into estrogen receptor function in human breast cancer. Ann Med. 2003;35:614–31.CrossRefPubMedGoogle Scholar
  41. 41.
    Hammond MEH, Hayes DF, Dowsett M, et al. American society of clinical oncology/college of american pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol. 2010;28:2784–95.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Helguero LA, Faulds MH, Gustafsson JA, Haldosen LA. Estrogen receptors alfa (ER alpha) and beta (ER beta) differentially regulate proliferation and apoptosis of the normal murine mammary epithelial cell line HC11. Oncogene. 2005;24:6605–16.CrossRefPubMedGoogle Scholar
  43. 43.
    Speirs V, Skliris GP, Burdall SE, Carder PJ. Distinct expression patterns of ER alpha and ER beta in normal human mammary gland. J Clin Pathol. 2002;55:371–4.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Palmieri C, Cheng GJ, Saji S, et al. Estrogen receptor beta in breast cancer. Endocr Relat Cancer. 2002;9:1–13.CrossRefPubMedGoogle Scholar
  45. 45.
    Forster C, Makela S, Warri A, et al. Involvement of estrogen receptor beta in terminal differentiation of mammary gland epithelium. Proc Natl Acad Sci USA. 2002;99:15578–83.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Feng Y, Manka D, Wagnert KU, Khan SA. Estrogen receptor-alpha expression in the mammary epithelium is required for ductal and alveolar morphogenesis in mice. Proc Natl Acad Sci USA. 2007;104:14718–23.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Bocchinfuso WP, Korach KS. Mammary gland development and tumorigenesis in estrogen receptor knockout mice. J Mammary Gland Biol Neoplasia. 1997;2:323–34.CrossRefPubMedGoogle Scholar
  48. 48.
    Hartman J, Muller P, Foster JS, Wimalasena J, Gustafsson JA, Strom A. HES-1 inhibits 17 beta-estradiol and heregulin-beta 1-mediated upregulation of E2F-1. Oncogene. 2004;23:8826–33.CrossRefPubMedGoogle Scholar
  49. 49.
    Warner M, Gustafsson JA. The role of estrogen receptor beta (ER beta) in malignant diseases: a new potential target for antiproliferative drugs in prevention and treatment of cancer. Biochem Biophys Res Commun. 2010;396:63–6.CrossRefPubMedGoogle Scholar
  50. 50.
    Williams C, Edvardsson K, Lewandowski SA, Strom A, Gustafsson JA. A genome-wide study of the repressive effects of estrogen receptor beta on estrogen receptor alpha signaling in breast cancer cells. Oncogene. 2008;27:1019–32.CrossRefPubMedGoogle Scholar
  51. 51.
    Yager JD, Davidson NE. Mechanisms of disease: estrogen carcinogenesis in breast cancer. N Engl J Med. 2006;354:270–82.CrossRefPubMedGoogle Scholar
  52. 52.
    Hao LK, Wang YJ, Duan YS, Bu SM. Effects of treadmill exercise training on liver fat accumulation and estrogen receptor alpha expression in intact and ovariectomized rats with or without estrogen replacement treatment. Eur J Appl Physiol. 2010;109:879–86.CrossRefPubMedGoogle Scholar
  53. 53.
    Rauf S, Soejono S, Partadiredja G. Effects of treadmill exercise training on cerebellar estrogen and estrogen receptors, serum estrogen, and motor coordination performance of ovariectomized rats. Iran J Basic Med Sci. 2015;18:587–92.PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Ana I. Faustino-Rocha
    • 1
    • 2
    • 3
    • 7
    Email author
  • Adelina Gama
    • 1
    • 3
  • Paula A. Oliveira
    • 1
    • 2
  • Antonieta Alvarado
    • 2
    • 4
  • Maria J. Neuparth
    • 5
    • 6
  • Rita Ferreira
    • 7
  • Mário Ginja
    • 1
    • 2
  1. 1.Department of Veterinary Sciences, School of Agrarian and Veterinary SciencesUniversity of Trás-os-Montes and Alto Douro, UTADVila RealPortugal
  2. 2.Center for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB)University of Trás-os-Montes and Alto Douro, UTADVila RealPortugal
  3. 3.Animal and Veterinary Research Center (CECAV)University of Trás-os-Montes and Alto Douro, UTADVila RealPortugal
  4. 4.Área de Patología, Decanato de Ciencias VeterinariasUniversidad Centroccidental “Lisandro Alvarado”, UCLALaraVenezuela
  5. 5.Institute of Research and Advanced Training in Health Sciences and Technologies (IINFACTS)CESPUGandraPortugal
  6. 6.Faculty of Sports, Research Centre in Physical Activity, Health and Leisure (CIAFEL)University of PortoPortoPortugal
  7. 7.Department of Chemistry, Organic Chemistry, Natural Products and Foodstuffs (QOPNA), Mass Spectrometry CenterUniversity of AveiroAveiroPortugal

Personalised recommendations