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Hypoxia pp 81-93 | Cite as

Evaluating the Metabolic Impact of Hypoxia on Pancreatic Cancer Cells

  • Divya Murthy
  • Enza Vernucci
  • Gennifer Goode
  • Jaime Abrego
  • Pankaj K. Singh
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1742)

Abstract

Hypoxia is frequently observed in human cancers and induces global metabolic reprogramming that includes an increase in glucose uptake and glycolysis, alterations in NAD(P)H/NAD(P)+ and intracellular ATP levels, and increased utilization of glutamine as the major precursor for fatty acid synthesis. In this chapter, we describe in detail various physiological assays that have been adopted to study the metabolic shift propagated by exposure to hypoxic conditions in pancreatic cell culture model that includes glucose uptake, glutamine uptake, and lactate release by pancreatic cancer cell lines. We have also elaborated the assays to evaluate the ratio of NAD(P)H/NAD(P)+ and intracellular ATP estimation using the commercially available kit to assess the metabolic state of cancer cells.

Key words

Hypoxia HIF Pancreatic cancer Cancer metabolism Glucose uptake Glutamine uptake Lactate release ATP estimation NADPH/NADP+ ratio 

References

  1. 1.
    Chaika NV, Gebregiworgis T, Lewallen ME, Purohit V, Radhakrishnan P, Liu X, Zhang B, Mehla K, Brown RB, Caffrey T, Yu F, Johnson KR, Powers R, Hollingsworth MA, Singh PK (2012) MUC1 mucin stabilizes and activates hypoxia-inducible factor 1 alpha to regulate metabolism in pancreatic cancer. Proc Natl Acad Sci U S A 109(34):13787–13792. https://doi.org/10.1073/pnas.1203339109 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Erickson LA, Highsmith WE Jr, Fei P, Zhang J (2015) Targeting the hypoxia pathway to treat pancreatic cancer. Drug Des Devel Ther 9:2029–2031. https://doi.org/10.2147/DDDT.S80888 PubMedPubMedCentralGoogle Scholar
  3. 3.
    Bertout JA, Patel SA, Simon MC (2008) The impact of O2 availability on human cancer. Nat Rev Cancer 8(12):967–975. https://doi.org/10.1038/nrc2540 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Kamiya Mehla, Pankaj K. Singh, (2014) MUC1: A novel metabolic master regulator. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1845(2):126–135Google Scholar
  5. 5.
    Surendra K. Shukla, Vinee Purohit, Kamiya Mehla, Venugopal Gunda, Nina V. Chaika, Enza Vernucci, Ryan J. King, Jaime Abrego, Gennifer D. Goode, Aneesha Dasgupta, Alysha L. Illies, Teklab Gebregiworgis, Bingbing Dai, Jithesh J. Augustine, Divya Murthy, Kuldeep S. Attri, Oksana Mashadova, Paul M. Grandgenett, Robert Powers, Quan P. Ly, Audrey J. Lazenby, Jean L. Grem, Fang Yu, José M. Matés, John M. Asara, Jung-whan Kim, Jordan H. Hankins, Colin Weekes, Michael A. Hollingsworth, Natalie J. Serkova, Aaron R. Sasson, Jason B. Fleming, Jennifer M. Oliveto, Costas A. Lyssiotis, Lewis C. Cantley, Lyudmyla Berim, Pankaj K. Singh, (2017) MUC1 and HIF-1alpha Signaling Crosstalk Induces Anabolic Glucose Metabolism to Impart Gemcitabine Resistance to Pancreatic Cancer. Cancer Cell 32(1):71–87.e7Google Scholar
  6. 6.
    Denko NC (2008) Hypoxia, HIF1 and glucose metabolism in the solid tumour. Nat Rev Cancer 8(9):705–713. https://doi.org/10.1038/nrc2468 CrossRefPubMedGoogle Scholar
  7. 7.
    Iyer NV, Kotch LE, Agani F, Leung SW, Laughner E, Wenger RH, Gassmann M, Gearhart JD, Lawler AM, Yu AY, Semenza GL (1998) Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev 12(2):149–162CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Chen C, Pore N, Behrooz A, Ismail-Beigi F, Maity A (2001) Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia. J Biol Chem 276(12):9519–9525. https://doi.org/10.1074/jbc.M010144200 CrossRefPubMedGoogle Scholar
  9. 9.
    Hansen PA, Gulve EA, Holloszy JO (1994) Suitability of 2-deoxyglucose for in vitro measurement of glucose transport activity in skeletal muscle. J Appl Physiol 76(2):979–985CrossRefPubMedGoogle Scholar
  10. 10.
    Abrego J, Gunda V, Vernucci E, Shukla SK, King RJ, Dasgupta A, Goode G, Murthy D, Yu F, Singh PK (2017) GOT1-mediated anaplerotic glutamine metabolism regulates chronic acidosis stress in pancreatic cancer cells. Cancer Lett 400:37–46. https://doi.org/10.1016/j.canlet.2017.04.029 CrossRefPubMedGoogle Scholar
  11. 11.
    Goode G, Gunda V, Chaika NV, Purohit V, Yu F, Singh PK (2017) MUC1 facilitates metabolomic reprogramming in triple-negative breast cancer. PloS one 12(5):e0176820. doi:10.1371/journal.pone.0176820Google Scholar
  12. 12.
    Surendra K. Shukla, Aneesha Dasgupta, Kamiya Mehla, Venugopal Gunda, Enza Vernucci, Joshua Souchek, Gennifer Goode, Ryan King, Anusha Mishra, Ibha Rai, Sangeetha Nagarajan, Nina V. Chaika, Fang Yu, Pankaj K. Singh, (2015) Silibinin-mediated metabolic reprogramming attenuates pancreatic cancer-induced cachexia and tumor growth. Oncotarget 6(38):41146–41161Google Scholar
  13. 13.
    Sun RC, Denko NC (2014) Hypoxic regulation of glutamine metabolism through HIF1 and SIAH2 supports lipid synthesis that is necessary for tumor growth. Cell Metab 19(2):285–292. https://doi.org/10.1016/j.cmet.2013.11.022 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Allison SJ, Knight JR, Granchi C, Rani R, Minutolo F, Milner J, Phillips RM (2014) Identification of LDH-A as a therapeutic target for cancer cell killing via (i) p53/NAD(H)-dependent and (ii) p53-independent pathways. Oncogene 3:e102. https://doi.org/10.1038/oncsis.2014.16 CrossRefGoogle Scholar
  15. 15.
    Henry RJ, Chiamori N, Golub OJ, Berkman S (1960) Revised spectrophotometric methods for the determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, and lactic acid dehydrogenase. Am J Clin Pathol 34:381–398CrossRefPubMedGoogle Scholar
  16. 16.
    Lloyd B, Burrin J, Smythe P, Alberti KG (1978) Enzymic fluorometric continuous-flow assays for blood glucose, lactate, pyruvate, alanine, glycerol, and 3-hydroxybutyrate. Clin Chem 24(10):1724–1729PubMedGoogle Scholar
  17. 17.
    Parks SK, Mazure NM, Counillon L, Pouyssegur J (2013) Hypoxia promotes tumor cell survival in acidic conditions by preserving ATP levels. J Cell Physiol 228(9):1854–1862. https://doi.org/10.1002/jcp.24346 CrossRefPubMedGoogle Scholar
  18. 18.
    Fan J, Kamphorst JJ, Mathew R, Chung MK, White E, Shlomi T, Rabinowitz JD (2013) Glutamine-driven oxidative phosphorylation is a major ATP source in transformed mammalian cells in both normoxia and hypoxia. Mol Syst Biol 9:712. https://doi.org/10.1038/msb.2013.65 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Divya Murthy
    • 1
  • Enza Vernucci
    • 1
  • Gennifer Goode
    • 1
  • Jaime Abrego
    • 1
  • Pankaj K. Singh
    • 1
    • 2
    • 3
    • 4
  1. 1.The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical CenterOmahaUSA
  2. 2.Department of Pathology and MicrobiologyUniversity of Nebraska Medical CenterOmahaUSA
  3. 3.Department of Genetics, Cell Biology and AnatomyUniversity of Nebraska Medical CenterOmahaUSA
  4. 4.Department of Biochemistry and Molecular BiologyUniversity of Nebraska Medical CenterOmahaUSA

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