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Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability

Stepanenko, A.A. ; Dmitrenko, V.V.

Gene, 2015-12, Vol.574 (2), p.193-203 [Periódico revisado por pares]

Netherlands: Elsevier B.V

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  • Título:
    Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability
  • Autor: Stepanenko, A.A. ; Dmitrenko, V.V.
  • Assuntos: Animals ; Biological Assay - methods ; Biological Assay - standards ; Cancer ; Cell Count - methods ; Cell Count - standards ; Cell Line, Tumor ; Cell Survival - drug effects ; Dacarbazine - analogs & derivatives ; Dacarbazine - pharmacology ; Drug resistance ; Female ; Formazans - pharmacology ; Glioblastoma ; Humans ; Inhibitory Concentration 50 ; Rapamycin ; Rats ; Rats, Wistar ; Sirolimus - analogs & derivatives ; Sirolimus - pharmacology ; Temozolomide ; Temsirolimus ; Tetrazolium Salts - pharmacology ; U0126
  • É parte de: Gene, 2015-12, Vol.574 (2), p.193-203
  • Notas: ObjectType-Article-1
    SourceType-Scholarly Journals-1
    ObjectType-Feature-2
    content type line 23
  • Descrição: The MTT assay (to a less degree MTS, XTT or WST) is a widely exploited approach for measuring cell viability/drug cytotoxicity. MTT reduction occurs throughout a cell and can be significantly affected by a number of factors, including metabolic and energy perturbations, changes in the activity of oxidoreductases, endo-/exocytosis and intracellular trafficking. Over/underestimation of cell viability by the MTT assay may be due to both adaptive metabolic and mitochondrial reprogramming of cells subjected to drug treatment-mediated stress and inhibitor off-target effects. Previously, imatinib, rottlerin, ursolic acid, verapamil, resveratrol, genistein nanoparticles and some polypeptides were shown to interfere with MTT reduction rate resulting in inconsistent results between the MTT assay and alternative assays. Here, to test the under/overestimation of viability by the MTT assay, we compared results derived from the MTT assay with the trypan blue exclusion assay after treatment of glioblastoma U251, T98G and C6 cells with three widely used inhibitors with the known direct and side effects on energy and metabolic homeostasis — temozolomide (TMZ), a DNA-methylating agent, temsirolimus (TEM), an inhibitor of mTOR kinase, and U0126, an inhibitor of MEK1/2 kinases. Inhibitors were applied shortly as in IC50 evaluating studies or long as in studies focusing on drug resistance acquisition. We showed that over/underestimation of cell viability by the MTT assay and its significance depends on a cell line, a time point of viability measurement and other experimental parameters. Furthermore, we provided a comprehensive survey of factors that should be accounted in the MTT assay. To avoid result misinterpretation, supplementation of the tetrazolium salt-based assays with other non-metabolic assays is recommended. •The metabolic and energy perturbations can significantly impact the MTT assay readout.•Temozolomide, temsirolimus and U0126 have effects on energy/metabolism homeostasis.•The MTT assay can over/underestimate viability of TMZ, TEM or U0126-treated cells.•Off-target effects are an unavoidable reality for majority of inhibitors.•To avoid delusion, the MTT assay should be supplemented with non-metabolic methods.
  • Editor: Netherlands: Elsevier B.V
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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