, 14:40 | Cite as

Untargeted and stable isotope-assisted metabolomic analysis of MDA-MB-231 cells under hypoxia

  • Jie Yang
  • Jianhua Cheng
  • Bo Sun
  • Haijing Li
  • Shengming Wu
  • Fangting Dong
  • Xianzhong Yan
Original Article



Hypoxia commonly occurs in cancers and is highly related with the occurrence, development and metastasis of cancer. Treatment of triple negative breast cancer remains challenge. Knowledge about the metabolic status of triple negative breast cancer cell lines in hypoxia is valuable for the understanding of molecular mechanisms of this tumor subtype to develop effective therapeutics.


Comprehensively characterize the metabolic profiles of triple negative breast cancer cell line MDA-MB-231 in normoxia and hypoxia and the pathways involved in metabolic changes in hypoxia.


Differences in metabolic profiles affected pathways of MDA-MB-231 cells in normoxia and hypoxia were characterized using GC–MS based untargeted and stable isotope assisted metabolomic techniques.


Thirty-three metabolites were significantly changed in hypoxia and nine pathways were involved. Hypoxia increased glycolysis, inhibited TCA cycle, pentose phosphate pathway and pyruvate carboxylation, while increased glutaminolysis in MDA-MB-231 cells.


The current results provide metabolic differences of MDA-MB-231 cells in normoxia and hypoxia conditions as well as the involved metabolic pathways, demonstrating the power of combined use of untargeted and stable isotope-assisted metabolomic methods in comprehensive metabolomic analysis.


Breast cancer Stable isotope-assisted metabolomics MDA-MB-231 GC-TOF MS Glycolysis Glutaminolysis 



This work was supported by Grants from the National Natural Science Foundation of China (81001419, 81273478), the National Science and Technology Major Project (2012ZX09301003-001-010).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Research involving animal and human rights

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

11306_2018_1338_MOESM1_ESM.docx (171 kb)
Supplementary Material 1 (DOCX 170 KB)
11306_2018_1338_MOESM2_ESM.xlsx (36 kb)
Supplementary Table 1 (XLSX 36 KB)
11306_2018_1338_MOESM3_ESM.xlsx (46 kb)
Supplementary Table 2 (XLSX 46 KB)


  1. Allen, E., Mieville, P., Warren, C. M., Saghafinia, S., Li, L., Peng, M. W., et al. (2016). Metabolic symbiosis enables adaptive resistance to anti-angiogenic therapy that is dependent on mTOR signaling. Cell Reports, 15(6), 1144–1160. Scholar
  2. Amelio, I., Cutruzzolá, F., Antonov, A., Agostini, M., & Melino, G. (2014). Serine and glycine metabolism in cancer. Trends in Biochemical Sciences, 39(4), 191–198. Scholar
  3. Armitage, E. G., Kotze, H. L., Allwood, J. W., Dunn, W. B., Goodacre, R., & Williams, K. J. (2015). Metabolic profiling reveals potential metabolic markers associated with Hypoxia Inducible Factor-mediated signalling in hypoxic cancer cells. Scientific Reports, 5, 15649. Scholar
  4. Cairns, R. A., Harris, I. S., & Mak, T. W. (2011). Regulation of cancer cell metabolism. Nature Reviews Cancer, 11(2), 85–95. Scholar
  5. Cairns, R. A., & Mak, T. W. (2016). The current state of cancer metabolism. Nature Reviews Cancer, 16, 613–614.CrossRefGoogle Scholar
  6. Camarda, R., Zhou, A. Y., Kohnz, R. A., Balakrishnan, S., Mahieu, C., Anderton, B., et al. (2016). Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer. Nature Medicine, 22(4), 427–432. Scholar
  7. Carlisle, S. M., Trainor, P. J., Yin, X., Doll, M. A., Stepp, M. W., States, J. C., et al. (2016). Untargeted polar metabolomics of transformed MDA-MB-231 breast cancer cells expressing varying levels of human arylamine N-acetyltransferase 1. Metabolomics, 12(7), 111. Scholar
  8. Carmeliet, P., Dor, Y., Herbert, J. M., Fukumura, D., Brusselmans, K., Dewerchin, M., et al. (1998). Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature, 394(6692), 485–490. Scholar
  9. Chavez, K. J., Garimella, S. V., & Lipkowitz, S. (2010). Triple negative breast cancer cell lines: One tool in the search for better treatment of triple negative breast cancer. Breast Disease, 32(1–2), 35–48. Scholar
  10. Chen, Y. J., Mahieu, N. G., Huang, X., Singh, M., Crawford, P. A., Johnson, S. L., et al. (2016). Lactate metabolism is associated with mammalian mitochondria. Nature Chemical Biology, 12(11), 937–943. Scholar
  11. Crown, J., O’Shaughnessy, J., & Gullo, G. (2012). Emerging targeted therapies in triple-negative breast cancer. Annals of Oncology, 23(Suppl 6), vi56–vi65.CrossRefPubMedGoogle Scholar
  12. Dang, L., White, D. W., Gross, S., Bennett, B. D., Bittinger, M. A., Driggers, E. M., et al. (2009). Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature, 462(7274), 739. Scholar
  13. de Lint, K., Poell, J. B., Soueidan, H., Jastrzebski, K., Vidal Rodriguez, J., Lieftink, C., et al. (2016). Sensitizing Triple-Negative Breast Cancer to PI3K Inhibition by Cotargeting IGF1R. Molecular Cancer Therapeutics, 15(7), 1545–1556.CrossRefPubMedGoogle Scholar
  14. Deng, W., Jiang, X., Mei, Y., Sun, J., Ma, R., Liu, X., et al. (2008). Role of ornithine decarboxylase in breast cancer. Acta Biochimica et Biophysica Sinica (Shanghai), 40(3), 235–243.CrossRefGoogle Scholar
  15. Elvidge, G. P., Glenny, L., Appelhoff, R. J., Ratcliffe, P. J., Ragoussis, J., & Gleadle, J. M. (2006). Concordant regulation of gene expression by hypoxia and 2-oxoglutarate-dependent dioxygenase inhibition: The role of HIF-1alpha, HIF-2alpha, and other pathways. Journal of Biological Chemistry, 281(22), 15215–15226. Scholar
  16. Frezza, C., Tennant, D. A., & Gottlieb, E. (2010). IDH1 mutations in gliomas: When an enzyme loses its grip. Cancer Cell, 17(1), 7–9. Scholar
  17. Gaglio, D., Metallo, C. M., Gameiro, P. A., Hiller, K., Danna, L. S., Balestrieri, C., et al. (2011). Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth. Molecular Systems Biology, 7, 523. Scholar
  18. Gameiro, P. A., Yang, J., Metelo, A. M., Perez-Carro, R., Baker, R., Wang, Z., et al. (2013). In vivo HIF-mediated reductive carboxylation is regulated by citrate levels and sensitizes VHL-deficient cells to glutamine deprivation. Cell Metabolism, 17(3), 372–385. Scholar
  19. Gao, X., Lin, S. H., Ren, F., Li, J. T., Chen, J. J., Yao, C. B., et al. (2016). Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia. Nature Communications, 7, 11960. Scholar
  20. Gleadle, J. M., & Ratcliffe, P. J. (1997). Induction of hypoxia-inducible factor-1, erythropoietin, vascular endothelial growth factor, and glucose transporter-1 by hypoxia: Evidence against a regulatory role for Src kinase. Blood, 89(2), 503–509.PubMedGoogle Scholar
  21. Gordan, J. D., Thompson, C. B., & Simon, M. C. (2007). HIF and c-Myc: Sibling rivals for control of cancer cell metabolism and proliferation. Cancer Cell, 12(2), 108–113. Scholar
  22. Harris, A. L. (2002). Hypoxia-a key regulatory factor in tumour growth. Nature Reviews Cancer, 2(1), 38–47. Scholar
  23. Huang, S., Chong, N., Lewis, N. E., Jia, W., Xie, G., & Garmire, L. X. (2016). Novel personalized pathway-based metabolomics models reveal key metabolic pathways for breast cancer diagnosis. Genome Medicine, 8(1), 34. Scholar
  24. Intlekofer, A. M., Dematteo, R. G., Venneti, S., Finley, L. W., Lu, C., Judkins, A. R., et al. (2015). Hypoxia induces production of L-2-hydroxyglutarate. Cell Metabolism, 22(2), 304–311. Scholar
  25. Kanaan, Y. M., Sampey, B. P., Beyene, D., Esnakula, A. K., Naab, T. J., Ricks-Santi, L. J., et al. (2014). Metabolic profile of triple-negative breast cancer in African-American women reveals potential biomarkers of aggressive disease. Cancer Genomics & Proteomics, 11(6), 279–294.Google Scholar
  26. Kim, J. W., Tchernyshyov, I., Semenza, G. L., & Dang, C. V. (2006). HIF-1-mediated expression of pyruvate dehydrogenase kinase: A metabolic switch required for cellular adaptation to hypoxia. Cell Metabolism, 3(3), 177–185. Scholar
  27. Kim, J. W., Zeller, K. I., Wang, Y., Jegga, A. G., Aronow, B. J., O’Donnell, K. A., et al. (2004). Evaluation of myc E-box phylogenetic footprints in glycolytic genes by chromatin immunoprecipitation assays. Molecular and Cellular Biology, 24(13), 5923–5936. Scholar
  28. Lane, A. N., Tan, J., Wang, Y., Yan, J., Higashi, R. M., & Fan, T. W. (2017). Probing the metabolic phenotype of breast cancer cells by multiple tracer stable isotope resolved metabolomics. Metabolic Engineering. Scholar
  29. Le, A., Lane, A. N., Hamaker, M., Bose, S., Gouw, A., Barbi, J., et al. (2012). Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells. Cell Metabolism, 15(1), 110–121. Scholar
  30. Lee, D. C., Sohn, H. A., Park, Z. Y., Oh, S., Kang, Y. K., Lee, K. M., et al. (2015). A lactate-induced response to hypoxia. Cell, 161(3), 595–609. Scholar
  31. Lehmann, B. D., Bauer, J. A., Chen, X., Sanders, M. E., Chakravarthy, A. B., Shyr, Y., et al. (2011). Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. Journal of Clinical Investigation, 121(7), 2750–2767.CrossRefPubMedPubMedCentralGoogle Scholar
  32. Li, X., Oprea-Ilies, G. M., & Krishnamurti, U. (2017). New developments in breast cancer and their impact on daily practice in pathology. Archives of Pathology & Laboratory Medicine, 141(4), 490–498.CrossRefGoogle Scholar
  33. Liedtke, C., Mazouni, C., Hess, K. R., André, F., Tordai, A., Mejia, J., et al. (2008). Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer. Journal of Clinical Oncology, 26(8), 1275–1281.CrossRefPubMedGoogle Scholar
  34. Lin, Y. Y., Cheng, W. B., & Wright, C. E. (1993). Glucose metabolism in mammalian cells as determined by mass isotopomer analysis. Analytical Biochemistry, 209(2), 267–273. Scholar
  35. Loffler, M., Carrey, E. A., & Zameitat, E. (2016). Orotate (orotic acid): An essential and versatile molecule. Nucleosides Nucleotides Nucleic Acids, 35(10–12), 566–577. Scholar
  36. Luo, W., Hu, H., Chang, R., Zhong, J., Knabel, M., O’Meally, R., et al. (2011). Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell, 145(5), 732–744. Scholar
  37. Mashimo, T., Pichumani, K., Vemireddy, V., Hatanpaa, K. J., Singh, D. K., Sirasanagandla, S., et al. (2014). Acetate is a bioenergetic substrate for human glioblastoma and brain metastases. Cell, 159(7), 1603–1614.CrossRefPubMedPubMedCentralGoogle Scholar
  38. Mathupala, S. P., Rempel, A., & Pedersen, P. L. (2001). Glucose catabolism in cancer cells: Identification and characterization of a marked activation response of the type II hexokinase gene to hypoxic conditions. Journal of Biological Chemistry, 276(46), 43407–43412. Scholar
  39. Metallo, C. M., Gameiro, P. A., Bell, E. L., Mattaini, K. R., Yang, J., Hiller, K., et al. (2012). Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature, 481(7381), 380–384. Scholar
  40. Nemoto, T., Hori, H., Yoshimoto, M., Seyama, Y., & Kubota, S. (2002). Overexpression of ornithine decarboxylase enhances endothelial proliferation by suppressing endostatin expression. Blood, 99(4), 1478–1481.CrossRefPubMedGoogle Scholar
  41. Pacold, M. E., Brimacombe, K. R., Chan, S. H., Rohde, J. M., Lewis, C. A., Swier, L. J., et al. (2016). A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate. Nature Chemical Biology, 12(6), 452–458. Scholar
  42. Pal, S. K., Childs, B. H., & Pegram, M. (2011). Triple negative breast cancer: Unmet medical needs. Breast Cancer Research and Treatment, 125(3), 627–636. Scholar
  43. Peterson, A. L., Walker, A. K., Sloan, E. K., & Creek, D. J. (2016). Optimized method for untargeted metabolomics analysis of MDA-MB-231 breast cancer cells. Metabolites. Scholar
  44. Phannasil, P., Ansari, I. H., El Azzouny, M., Longacre, M. J., Rattanapornsompong, K., Burant, C. F., et al. (2017). Mass spectrometry analysis shows the biosynthetic pathways supported by pyruvate carboxylase in highly invasive breast cancer cells. Biochimica et Biophysica Acta, 1863(2), 537–551. Scholar
  45. Pollard, P. J., Briere, J. J., Alam, N. A., Barwell, J., Barclay, E., Wortham, N. C., et al. (2005). Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations. Human Molecular Genetics, 14(15), 2231–2239. Scholar
  46. Polyak, K. (2011). Heterogeneity in breast cancer. Journal of Clinical Investigation, 121(10), 3786–3788.CrossRefPubMedPubMedCentralGoogle Scholar
  47. Possemato, R., Marks, K. M., Shaul, Y. D., Pacold, M. E., Kim, D., Birsoy, K., et al. (2011). Functional genomics reveal that the serine synthesis pathway is essential in breast cancer. Nature, 476(7360), 346–350. Scholar
  48. Samanta, D., Gilkes, D. M., Chaturvedi, P., Xiang, L., & Semenza, G. L. (2014). Hypoxia-inducible factors are required for chemotherapy resistance of breast cancer stem cells. Proceedings of the National Academy of Sciences of the USA, 111(50), E5429–E5438. Scholar
  49. Semenza, G. L., Roth, P. H., Fang, H. M., & Wang, G. L. (1994). Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. Journal of Biological Chemistry, 269(38), 23757–23763.PubMedGoogle Scholar
  50. Shah, S. P., Roth, A., Goya, R., Oloumi, A., Ha, G., Zhao, Y., et al. (2012). The clonal and mutational evolution spectrum of primary triple-negative breast cancers. Nature, 486(7403), 395–399.PubMedGoogle Scholar
  51. Sonveaux, P., Vegran, F., Schroeder, T., Wergin, M. C., Verrax, J., Rabbani, Z. N., et al. (2008). Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. Journal of Clinical Investigation, 118(12), 3930–3942. Scholar
  52. Stincone, A., Prigione, A., Cramer, T., Wamelink, M. M. C., Campbell, K., Cheung, E., et al. (2015). The return of metabolism: Biochemistry and physiology of the pentose phosphate pathway. Biological Reviews of the Cambridge Philosophical Society, 90(3), 927–963. Scholar
  53. Sullivan, L. B., Gui, D. Y., & Vander Heiden, M. G. (2016). Altered metabolite levels in cancer: Implications for tumour biology and cancer therapy. Nature Reviews Cancer, 16(11), 680–693. Scholar
  54. Sun, R. C., & Denko, N. C. (2014). Hypoxic regulation of glutamine metabolism through HIF1 and SIAH2 supports lipid synthesis that is necessary for tumor growth. Cell Metabolism, 19(2), 285–292. Scholar
  55. Svensson, K. J., Welch, J. E., Kucharzewska, P., Bengtson, P., Bjurberg, M., Pahlman, S., et al. (2008). Hypoxia-mediated induction of the polyamine system provides opportunities for tumor growth inhibition by combined targeting of vascular endothelial growth factor and ornithine decarboxylase. Cancer Research, 68(22), 9291–9301. Scholar
  56. Torre, L. A., Bray, F., Siegel, R. L., Ferlay, J., Lortet-Tieulent, J., & Jemal, A. (2015). Global cancer statistics, 2012. CA, 65(2), 87–108. Scholar
  57. Tsai, I. L., Kuo, T. C., Ho, T. J., Harn, Y. C., Wang, S. Y., Fu, W. M., et al. (2013). Metabolomic dynamic analysis of hypoxia in MDA-MB-231 and the comparison with inferred metabolites from transcriptomics data. Cancers (Basel), 5(2), 491–510. Scholar
  58. Valli, A., Rodriguez, M., Moutsianas, L., Fischer, R., Fedele, V., Huang, H. L., et al. (2015). Hypoxia induces a lipogenic cancer cell phenotype via HIF1alpha-dependent and -independent pathways. Oncotarget, 6(4), 1920–1941. Scholar
  59. Vander Heiden, M. G., Locasale, J. W., Swanson, K. D., Sharfi, H., Heffron, G. J., Amador-Noguez, D., et al. (2010). Evidence for an alternative glycolytic pathway in rapidly proliferating cells. Science, 329(5998), 1492–1499. Scholar
  60. Weljie, A. M., Bondareva, A., Zang, P., & Jirik, F. R. (2011). (1)H NMR metabolomics identification of markers of hypoxia-induced metabolic shifts in a breast cancer model system. Journal of Biomolecular NMR, 49(3–4), 185–193. Scholar
  61. Willmann, L., Schlimpert, M., Halbach, S., Erbes, T., Stickeler, E., & Kammerer, B. (2015). Metabolic profiling of breast cancer: Differences in central metabolism between subtypes of breast cancer cell lines. Journal of Chromatography B, 1000, 95–104. Scholar
  62. Wood, T. (1986). Physiological functions of the pentose phosphate pathway. Cell Biochemistry & Function, 4(4), 241–247. Scholar
  63. Xia, J., & Wishart, D. S. (2011). Web-based inference of biological patterns, functions and pathways from metabolomic data using MetaboAnalyst. Nature Protocols, 6(6), 743–760. Scholar
  64. Yang, L., Moss, T., Mangala, L. S., Marini, J., Zhao, H., Wahlig, S., et al. (2014). Metabolic shifts toward glutamine regulate tumor growth, invasion and bioenergetics in ovarian cancer. Molecular Systems Biology, 10, 728. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.National Center of Biomedical AnalysisBeijingChina

Personalised recommendations