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Bioassays for Anticancer Activities

  • Janice McCauley
  • Ana Zivanovic
  • Danielle Skropeta
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1055)

Abstract

The MTT/MTS in vitro cell proliferation assay is one of the most widely used assays for evaluating preliminary anticancer activity of both synthetic derivatives and natural products and natural product extracts. The highly reliable, colorimetric based assay is readily performed on a wide range of cell lines. This assay gives an indication of whole cell cytotoxicity; however, to determine the exact molecular target further assays need to be performed. Of these, kinase inhibition assays are also one of the most widespread enzyme inhibition screening assays performed. Kinases are enzymes that play a key role in a number of physiological processes and their inhibitors have been found to exhibit anticancer activity against various human cancer cell lines. Herein, we describe the methods for performing both in vitro MTT/MTS cytotoxicity and kinase enzyme inhibition assays. These are two of the most useful anticancer screening techniques available that are relatively economical and can be easily and routinely performed in the laboratory to characterize anticancer activity. Both assays are highly versatile and can be modified to test against targeted disease processes by using specific kinase enzymes or cell lines.

Key words

MTS/MTT assays Cytotoxicity Anticancer activity Human cancer cell lines Enzyme inhibition Kinases 

Notes

Acknowledgment

This work was supported by the Center of Medicinal Chemistry and the School of Chemistry, University of Wollongong.

References

  1. 1.
    Almeida CA (2010) Cancer: basic science and clinical aspects. Wiley-Blackwell, LondonGoogle Scholar
  2. 2.
    Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70PubMedCrossRefGoogle Scholar
  3. 3.
    Altman R, Sarg M (2000) The cancer dictionary. Facts on File, New York, NYGoogle Scholar
  4. 4.
    Berg K, Zhai L, Chen M et al (1994) The use of watersoluble formazan complex to quantitate the cell number and mitochondrial function of Leishmania major promastigotes. Parasitol Res 80:235–239PubMedCrossRefGoogle Scholar
  5. 5.
    Malich G, Markovic B, Winder C (1997) The sensitivity and specificity of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines. Toxicology 124:179–192PubMedCrossRefGoogle Scholar
  6. 6.
    Matesic L, Locke JM, Bremner JB et al (2008) N-phenethyl and N-naphthylmethyl isatins and analogues as in vitro cytotoxic agents. Bioorg Med Chem 16:3118–3124PubMedCrossRefGoogle Scholar
  7. 7.
    Boyd MR, Paull KD (1995) Some practical considerations and applications of the National Cancer Institue in vitro anticancer drug discovery screen. Drug Dev Res 34:91–109CrossRefGoogle Scholar
  8. 8.
    Shoemaker RH (2006) The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer 6:813–823PubMedCrossRefGoogle Scholar
  9. 9.
    Collins JM (2012) Developmental therapeutics program NCI/NIH. http://dtp.nci.nih.gov. Accessed 29 Jun 2012
  10. 10.
    von Ahsen O, Bomer U (2005) High-throughput screening for kinase inhibitors. Chembiochem 6:481–490CrossRefGoogle Scholar
  11. 11.
    Copeland RA (ed) (2005) Evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists (Methods in biochemical analysis). Methods of biochemical analysis. Wiley, Hoboken, NJGoogle Scholar
  12. 12.
    Manning G, Whyte DB, Martinez R et al (2002) The protein kinase complement of the human genome. Science 298:1912–1934PubMedCrossRefGoogle Scholar
  13. 13.
    Goldstein D, Gray N, Zarrinkar P (2008) High-throughput kinase profiling as a platform for drug discovery. Nat Rev Drug Discov 7:391–397PubMedCrossRefGoogle Scholar
  14. 14.
    Liao JJL (2007) Molecular recognition of protein kinase binding pockets for design of potent and selective kinase inhibitors. J Med Chem 50:409–424PubMedCrossRefGoogle Scholar
  15. 15.
    Sharma P, Sharma R, Tyagi R (2008) Inhibitors of cyclin dependent kinases: useful targets for cancer treatment. Curr Cancer Drug Targets 8:53–75PubMedCrossRefGoogle Scholar
  16. 16.
    Skropeta D, Pastro N, Zivanovic A (2011) Kinase inhibitors from marine sponges. Mar Drugs 9:2131–2154PubMedCrossRefGoogle Scholar
  17. 17.
    Ono-Saito N, Niki I, Hidaka H (1999) H-series protein kinase inhibitors and potential clinical applications. Pharmacol Ther 82:123–131PubMedCrossRefGoogle Scholar
  18. 18.
    Shabb JB (2001) Physiological substrates of cAMP-dependent protein kinase. Chem Rev 101:2381–2412PubMedCrossRefGoogle Scholar
  19. 19.
    Chen AE, Ginty DD, Fan CM (2005) Protein kinase A signalling via CREB controls myogenesis induced by Wnt proteins. Nat Rev Drug Discov 433:317–322CrossRefGoogle Scholar
  20. 20.
    Tasken K, Aandahl EM (2004) Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiol Rev 84:137–167PubMedCrossRefGoogle Scholar
  21. 21.
    Arnsten AFT, Ramos BP, Birnbaum SG et al (2005) Protein kinase A as a therapeutic target for memory disorders: rationale and challenges. Trends Mol Med 11:121–128PubMedCrossRefGoogle Scholar
  22. 22.
    Suzuki M, Shinohara F, Endo M et al (2009) Zebularine suppresses the apoptotic potential of 5-fluorouracil via cAMP/PKA/CREB pathway against human oral squamous cell carcinoma cells. Cancer Chemother Pharmacol 64:223–232PubMedCrossRefGoogle Scholar
  23. 23.
    Putz T, Culig Z, Eder IE et al (1999) Epidermal growth factor (EGF) receptor blockade inhibits the action of EGF, insulin-like growth factor I, and a protein kinase A activator on the mitogen-activated protein kinase pathway in prostate cancer cell lines. Cancer Res 59:227–233PubMedGoogle Scholar
  24. 24.
    Bryn T, Mahic M, Aandahl EM et al (2008) Inhibition of protein kinase A improves effector function of monocytes from HIV-infected patients. AIDS Res Hum Retroviruses 24:1013–1015PubMedCrossRefGoogle Scholar
  25. 25.
    Torgersen KM, Vang T, Abrahamsen H et al (2002) Molecular mechanisms for protein kinase A-mediated modulation of immune function. Cell Signal 14:1–9PubMedCrossRefGoogle Scholar
  26. 26.
    Indira CV, Matesic L, Locke JM et al (2012) Anti-cancer activity of an acid-labile N-alkylisatin conjugate targeting the transferrin receptor. Cancer Lett 316:151–156CrossRefGoogle Scholar
  27. 27.
    Zivanovic A, Pastro NJ, Fromont J et al (2011) Kinase inhibitory, haemolytic and cytotoxic activity of three deep-water sponges from North Western Australia and their fatty acid composition. Nat Prod Commun 6:1921–1924PubMedGoogle Scholar
  28. 28.
    Promega (2009) Kinase-Glo® luminescent kinase assay platformGoogle Scholar
  29. 29.
    Baki A, Bielik A, Molnar L et al (2007) A high through-put luminsescent assay for glycogen synthase kinase-3 beta inhibitors. Assay Drug Dev Technol 5:75–83PubMedCrossRefGoogle Scholar
  30. 30.
    Promega (2012) CellTiter 96® AQueous one solution cell proliferation assay. pp 1–13Google Scholar
  31. 31.
    PromegaCorporation (2012) Promega protocols. http://www.promega.com/resources/protocols. Accessed 26 Jul 2012
  32. 32.
    Moore GE, Woods LK (1967) Culture media for human cells – RPMI 1603, RPMI 1634, RPMI 1640 and GEM 1717. Tissue Cult Assn 3:503–508CrossRefGoogle Scholar
  33. 33.
    Moore GE, Gerner RE, Franklin HA (1967) Culture of normal human leukocytes. J Am Med Assoc 199:519–524CrossRefGoogle Scholar
  34. 34.
    Koresawa M, Okabe T (2004) High-throughput screening with quantitation of ATP consumption: a universal non-radioisotope, homogeneous assay for protein kinase. Assay Drug Dev Technol 2:153–160PubMedCrossRefGoogle Scholar
  35. 35.
    Kashem MA, Nelson RM, Yingling JD et al (2007) Three mechanistically distinct kinase assays compared: measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors. J Biomol Screen 12:70–83PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Janice McCauley
    • 1
    • 2
  • Ana Zivanovic
    • 1
    • 3
  • Danielle Skropeta
    • 1
    • 3
  1. 1.School of ChemistryUniversity of WollongongWollongongAustralia
  2. 2.Shoalhaven Marine and Freshwater Centre, School of Biological SciencesUniversity of WollongongWollongongAustralia
  3. 3.Centre for Medicinal ChemistryUniversity of WollongongWollongongAustralia

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