Stem Cell Reviews and Reports

, Volume 12, Issue 3, pp 340–351 | Cite as

Disparate Response to Methotrexate in Stem Versus Non-Stem Cells

  • Olivia S. Beane
  • Louise E. O. Darling
  • Vera C. Fonseca
  • Eric M. DarlingEmail author


Methotrexate (MTX) is a commonly used chemotherapeutic agent that kills cancer cells by binding dihydrofolate reductase (DHFR) as a competitive inhibitor. Due to its non-selectivity, MTX also impairs normal (non-cancerous) cell function and causes long-term damage to healthy tissue. These consequences have been investigated extensively in bone-derived cells due to their sensitivity to the drug. While DHFR likely plays a role in normal cell response to MTX, research in this area is limited. Moreover, how MTX sensitivity differs among cell types responsible for maintaining connective tissues is unknown. The goal of this study was to investigate the role of DHFR and subsequent nucleotide synthesis in normal cell response to MTX. We also sought to compare adverse effects of MTX among normal cell types to identify sensitive populations and resistant cell sources for regenerative procedures targeting patients undergoing chemotherapy. DHFR overexpression or exogenous amino acid + nucleoside delivery rescued normal cells from adverse MTX effects. Conversely, DHFR knockdown impaired MTX-treated adipose-derived stem cell (ASC) osteogenesis. Proliferation of ASCs and bone marrow stem cells was more resistant to MTX than that of terminally differentiated osteoblasts. However, stem cells became susceptible to the drug after beginning differentiation. These results suggest that the ability of stem cells to survive and to maintain their surrounding tissues likely depends on whether they are in a “stem” state when exposed to MTX. Therapeutic strategies that delay the differentiation of stem cells until clearance of the drug may produce more favorable outcomes in the long-term health of treated tissues.


Cancer Chemotherapy Mesenchymal stem cell Methotrexate Regenerative medicine 



The authors would like to thank Jason Machan for advice on statistical analyses, Paul Liu and Mikki Ciombor for providing lipoaspirate samples, and Mark Dooner and the COBRE Flow Cytometry Core at Rhode Island Hospital for their assistance with FACS experiments. This work was supported by awards from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR063642; EMD), National Science Foundation (CAREER Award, CBET1253189; EMD), and a Staley Small Grant from Wellesley College (LEOD). The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or National Science Foundation.

Author Contributions

OSB, LEOD, and EMD designed the study and wrote the manuscript. OSB conducted all cell culture, Western blots, PCR, and assays for proliferation and differentiation. LEOD conducted all plasmid synthesis work. VCF assisted with histological staining.

Compliance with Ethical Standards

Conflict of Interest

The authors declare no potential conflict of interest.

Supplementary material

12015_2016_9645_Fig9_ESM.gif (13 kb)
Supplemental Figure 1

Effects of DHFR knockdown on ASC osteogenesis. On a per cell basis, MTX had no significant effect on ASC ALP activity. However, DHFR knockdown reduced ALP activity overall compared to control siRNA (p < 0.05), but no individual comparisons showed significance. ALP activity is presented as a percent of untreated values within control siRNA conditions. Error bars depict standard deviation. (GIF 12 kb)

12015_2016_9645_MOESM1_ESM.tif (836 kb)
High resolution image (TIFF 835 kb)


  1. 1.
    Wisnivesky, J. P., Smith, C. B., Packer, S., et al. (2011). Survival and risk of adverse events in older patients receiving postoperative adjuvant chemotherapy for resected stages II-IIIA lung cancer: observational cohort study. BMJ, 343, d4013.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Berry, D. A., Cronin, K. A., Plevritis, S. K., et al. (2005). Effect of screening and adjuvant therapy on mortality from breast cancer. The New England Journal of Medicine, 353, 1784–1792.CrossRefPubMedGoogle Scholar
  3. 3.
    Saltz, L. B., & Kemeny, N. E. (1996). Adjuvant chemotherapy of colorectal cancer. The Oncologist, 1, 22–29.PubMedGoogle Scholar
  4. 4.
    Gerber, D. E. (2008). Targeted therapies: a new generation of cancer treatments. American Family Physician, 77, 311–319.PubMedGoogle Scholar
  5. 5.
    Deep, G., & Agarwal, R. (2008). New combination therapies with cell-cycle agents. Current Opinion in Investigational Drugs, 9, 591–604.PubMedPubMedCentralGoogle Scholar
  6. 6.
    Davies, J. H., Evans, B. A., Jenney, M. E., & Gregory, J. W. (2003). Effects of chemotherapeutic agents on the function of primary human osteoblast-like cells derived from children. The Journal of Clinical Endocrinology and Metabolism, 88, 6088–6097.CrossRefPubMedGoogle Scholar
  7. 7.
    Baum, B. J., Bodner, L., Fox, P. C., Izutsu, K. T., Pizzo, P. A., & Wright, W. E. (1985). Therapy-induced dysfunction of salivary glands: implications for oral health. Special Care in Dentistry: Official Publication of the American Association of Hospital Dentists, the Academy of Dentistry for the Handicapped, and the American Society for Geriatric Dentistry, 5, 274–277.CrossRefGoogle Scholar
  8. 8.
    Cayley, P. J., Dunn, S. M., & King, R. W. (1981). Kinetics of substrate, coenzyme, and inhibitor binding to Escherichia coli dihydrofolate reductase. Biochemistry, 20, 874–879.CrossRefPubMedGoogle Scholar
  9. 9.
    Chabner, B. A., Allegra, C. J., Curt, G. A., et al. (1985). Polyglutamation of methotrexate. Is methotrexate a prodrug? The Journal of Clinical Investigation, 76, 907–912.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Chan, E. S., & Cronstein, B. N. (2002). Molecular action of methotrexate in inflammatory diseases. Arthritis Research, 4, 266–273.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Rivera, G. K., Raimondi, S. C., Hancock, M. L., et al. (1991). Improved outcome in childhood acute lymphoblastic leukaemia with reinforced early treatment and rotational combination chemotherapy. Lancet, 337, 61–66.CrossRefPubMedGoogle Scholar
  12. 12.
    Early Breast Cancer Trialists' Collaborative G., Peto R., Davies C., et al. (2012). Comparisons between different polychemotherapy regimens for early breast cancer: meta-analyses of long-term outcome among 100,000 women in 123 randomised trials. Lancet, 379, 432–444.Google Scholar
  13. 13.
    Choi, H. K., Hernan, M. A., Seeger, J. D., Robins, J. M., & Wolfe, F. (2002). Methotrexate and mortality in patients with rheumatoid arthritis: a prospective study. Lancet, 359, 1173–1177.CrossRefPubMedGoogle Scholar
  14. 14.
    DeOliveira, C. C., Acedo, S. C., Gotardo, E. M., et al. (2012). Effects of methotrexate on inflammatory alterations induced by obesity: an in vivo and in vitro study. Molecular and Cellular Endocrinology, 361, 92–98.CrossRefPubMedGoogle Scholar
  15. 15.
    Minaur, N. J., Jefferiss, C., Bhalla, A. K., & Beresford, J. N. (2002). Methotrexate in the treatment of rheumatoid arthritis. I. In vitro effects on cells of the osteoblast lineage. Rheumatology, 41, 735–740.CrossRefPubMedGoogle Scholar
  16. 16.
    Georgiou, K. R., Scherer, M. A., Fan, C. M., et al. (2012). Methotrexate chemotherapy reduces osteogenesis but increases adipogenic potential in the bone marrow. Journal of Cellular Physiology, 227, 909–918.CrossRefPubMedGoogle Scholar
  17. 17.
    Cohen, I. J., & Wolff, J. E. (2014). How long can folinic acid rescue be delayed after high-dose methotrexate without toxicity? Pediatric Blood & Cancer, 61, 7–10.CrossRefGoogle Scholar
  18. 18.
    Howell, S. B., Ensminger, W. D., Krishan, A., & Frei, E. (1978). Thymidine rescue of high-dose methotrexate in humans. Cancer Research, 38, 325–330.PubMedGoogle Scholar
  19. 19.
    Zuk, P. A., Zhu, M., Ashjian, P., et al. (2002). Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell, 13, 4279–4295.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Gimble, J. M., Katz, A. J., & Bunnell, B. A. (2007). Adipose-derived stem cells for regenerative medicine. Circulation Research, 100, 1249–1260.CrossRefPubMedGoogle Scholar
  21. 21.
    Spalding, K. L., Arner, E., Westermark, P. O., et al. (2008). Dynamics of fat cell turnover in humans. Nature, 453, 783–787.CrossRefPubMedGoogle Scholar
  22. 22.
    Chen, L., Hou, J., Ye, L., et al. (2014). MicroRNA-143 regulates adipogenesis by modulating the MAP2K5-ERK5 signaling. Scientific Reports, 4, 3819.PubMedPubMedCentralGoogle Scholar
  23. 23.
    Suga, H., Glotzbach, J. P., Sorkin, M., Longaker, M. T., & Gurtner, G. C. (2014). Paracrine mechanism of angiogenesis in adipose-derived stem cell transplantation. Annals of Plastic Surgery, 72, 234–241.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Beane, O. S., Fonseca, V. C., & Darling, E. M. (2014). Adipose-derived stem cells retain their regenerative potential after methotrexate treatment. Experimental Cell Research, 327, 222–233.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Qi, Z., Zhang, Y. M., Liu, L., Guo, X., Qin, J., & Cui, G. H. (2012). Mesenchymal stem cells derived from different origins have unique sensitivities to different chemotherapeutic agents. Cell Biology International, 36, 857–862.CrossRefPubMedGoogle Scholar
  26. 26.
    Estes, B. T., Diekman, B. O., Gimble, J. M., & Guilak, F. (2010). Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nature Protocols, 5, 1294–1311.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Estes, B. T., Diekman, B. O., & Guilak, F. (2008). Monolayer cell expansion conditions affect the chondrogenic potential of adipose-derived stem cells. Biotechnology and Bioengineering, 99, 986–995.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Youssef, J., Nurse, A. K., Freund, L. B., & Morgan, J. R. (2011). Quantification of the forces driving self-assembly of three-dimensional microtissues. Proceedings of the National Academy of Sciences of the United States of America, 108, 6993–6998.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Costa, H. J. Z. R., Bento, R. F., Salomone, R., et al. (2013). Mesenchymal bone marrow stem cells within polyglycolic acid tube observed in vivo after six weeks enhance facial nerve regeneration. Brain Research, 1510, 10–21.CrossRefPubMedGoogle Scholar
  30. 30.
    Zacharias, D. A., Violin, J. D., Newton, A. C., & Tsien, R. Y. (2002). Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science, 296, 913–916.CrossRefPubMedGoogle Scholar
  31. 31.
    Rosenblatt, D. S., Whitehead, V. M., Matiaszuk, N. V., Pottier, A., Vuchich, M. J., & Beaulieu, D. (1982). Differential effects of folinic acid and glycine, adenosine, and thymidine as rescue agents in methotrexate-treated human cells in relation to the accumulation of methotrexate polyglutamates. Molecular Pharmacology, 21, 718–722.PubMedGoogle Scholar
  32. 32.
    Li, J., Law, H. K. W., Lau, Y. L., & Chan, G. C. F. (2004). Differential damage and recovery of human mesenchymal stem cells after exposure to chemotherapeutic agents. Brit J Haematol, 127, 326–334.CrossRefGoogle Scholar
  33. 33.
    Schmittgen, T. D., & Livak, K. J. (2008). Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols, 3, 1101–1108.CrossRefPubMedGoogle Scholar
  34. 34.
    Zheng, B., Cao, B., Li, G., & Huard, J. (2006). Mouse adipose-derived stem cells undergo multilineage differentiation in vitro but primarily osteogenic and chondrogenic differentiation in vivo. Tissue Engineering, 12, 1891–1901.CrossRefPubMedGoogle Scholar
  35. 35.
    Labriola, N. R., & Darling, E. M. (2015). Temporal heterogeneity in single-cell gene expression and mechanical properties during adipogenic differentiation. Journal of Biomechanics, 48, 1058–1066.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Guilak, F., Lott, K. E., Awad, H. A., et al. (2006). Clonal analysis of the differentiation potential of human adipose-derived adult stem cells. Journal of Cellular Physiology, 206, 229–237.CrossRefPubMedGoogle Scholar
  37. 37.
    Reichert, J. C., Quent, V. M. C., Burke, L. J., Stansfield, S. H., Clements, J. A., & Hutmacher, D. W. (2010). Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment. Biomaterials, 31, 7928–7936.CrossRefPubMedGoogle Scholar
  38. 38.
    Arnoldo, A., Kittanakom, S., Heisler, L. E., et al. (2014). A genome scale overexpression screen to reveal drug activity in human cells. Genome Medicine, 6, 32.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Uchiyama, H., Sowa, Y., Wakada, M., et al. (2010). Cyclin-dependent kinase inhibitor SU9516 enhances sensitivity to methotrexate in human T-cell leukemia jurkat cells. Cancer Science, 101, 728–734.CrossRefPubMedGoogle Scholar
  40. 40.
    Kim, T. K., & Eberwine, J. H. (2010). Mammalian cell transfection: the present and the future. Analytical and Bioanalytical Chemistry, 397, 3173–3178.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Kimura, E., Nishimura, K., Sakata, K., Oga, S., Kashiwagi, K., & Igarashi, K. (2004). Methotrexate differentially affects growth of suspension and adherent cells. The International Journal of Biochemistry & Cell Biology, 36, 814–825.CrossRefGoogle Scholar
  42. 42.
    Schwartz, P. M., Barnett, S. K., Atillasoy, E. S., & Milstone, L. M. (1992). Methotrexate induces differentiation of human keratinocytes. Proceedings of the National Academy of Sciences of the United States of America, 89, 594–598.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Jolivet, J., Schilsky, R. L., Bailey, B. D., Drake, J. C., & Chabner, B. A. (1982). Synthesis, retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells. The Journal of Clinical Investigation, 70, 351–360.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Kennedy, D. G., Clarke, R., van den Berg, H. W., & Murphy, R. F. (1983). The kinetics of methotrexate polyglutamate formation and efflux in a human breast cancer cell line (MDA.MB.436): the effect of insulin. Biochemical Pharmacology, 32, 41–46.CrossRefPubMedGoogle Scholar
  45. 45.
    Piper, A. A., Nott, S. E., Mackinnon, W. B., & Tattersall, M. H. (1983). Critical modulation by thymidine and hypoxanthine of sequential methotrexate-5-fluorouracil synergism in murine L1210 cells. Cancer Research, 43, 5101–5105.PubMedGoogle Scholar
  46. 46.
    Bianchi, P. A. (1962). Thymidine phosphorylation and deoxyribonucleic acid synthesis in human leukaemic cells. Biochimica et Biophysica Acta, 55, 547–549.CrossRefPubMedGoogle Scholar
  47. 47.
    Dubbs, D. R., & Kit, S. (1964). Effect of halogenated pyrimidines and thymidine on growth of L-cells and a subline lacking thymidine kinase. Experimental Cell Research, 33, 19–28.CrossRefPubMedGoogle Scholar
  48. 48.
    Pinedo, H. M., Zaharko, D. S., Bull, J. M., & Chabner, B. A. (1976). The reversal of methotrexate cytotoxicity to mouse bone marrow cells by leucovorin and nucleosides. Cancer Research, 36, 4418–4424.PubMedGoogle Scholar
  49. 49.
    Wolfrom, C., Hepp, R., Hartmann, R., Breithaupt, H., & Henze, G. (1990). Pharmacokinetic study of methotrexate, folinic acid and their serum metabolites in children treated with high-dose methotrexate and leucovorin rescue. European Journal of Clinical Pharmacology, 39, 377–383.CrossRefPubMedGoogle Scholar
  50. 50.
    May, K. P., Mercill, D., McDermott, M. T., & West, S. G. (1996). The effect of methotrexate on mouse bone cells in culture. Arthritis and Rheumatism, 39, 489–494.CrossRefPubMedGoogle Scholar
  51. 51.
    Colak, S., & Medema, J. P. (2014). Human colonic fibroblasts regulate stemness and chemotherapy resistance of colon cancer stem cells. Cell Cycle, 0.Google Scholar
  52. 52.
    VanderVeen, M. J., Scheven, B. A. A., VanRoy, J. L. A. M., Damen, C. A., Lafeber, F. P. J. G., & Bijlsma, J. W. J. (1996). In vitro effects of methotrexate on human articular cartilage and bone-derived osteoblasts. Brit J Rheumatol, 35, 342–349.CrossRefGoogle Scholar
  53. 53.
    Uehara, R., Suzuki, Y., & Ichikawa, Y. (2001). Methotrexate (MTX) inhibits osteoblastic differentiation in vitro: possible mechanism of MTX osteopathy. The Journal of Rheumatology, 28, 251–256.PubMedGoogle Scholar
  54. 54.
    Georgiou, K. R., King, T. J., Scherer, M. A., Zhou, H., Foster, B. K., & Xian, C. J. (2012). Attenuated Wnt/beta-catenin signalling mediates methotrexate chemotherapy-induced bone loss and marrow adiposity in rats. Bone, 50, 1223–1233.CrossRefPubMedGoogle Scholar
  55. 55.
    Prochazka, E., Soukup, T., Hroch, M., et al. (2010). Methotrexate released in vitro from bone cement inhibits human stem cell proliferation in S/G2 phase. International Orthopaedics, 34, 137–142.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Olivia S. Beane
    • 1
  • Louise E. O. Darling
    • 2
  • Vera C. Fonseca
    • 3
  • Eric M. Darling
    • 1
    • 3
    • 4
    • 5
    Email author
  1. 1.Center for Biomedical EngineeringBrown UniversityProvidenceUSA
  2. 2.Department of Biological SciencesWellesley CollegeWellesleyUSA
  3. 3.Department of Molecular Pharmacology, Physiology, & BiotechnologyBrown UniversityProvidenceUSA
  4. 4.School of EngineeringBrown UniversityProvidenceUSA
  5. 5.Department of OrthopaedicsBrown UniversityProvidenceUSA

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