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Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers
Rottlerin is a natural product isolated from Mallotus philippinensis. This polyphenolic compound, originally described as a selective inhibitor of PKCδ, can inhibit many other PKC-unrelated kinases and has a number of biological actions, including mitochondrial uncoupling effects. We recently found...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2009
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056051/ https://www.ncbi.nlm.nih.gov/pubmed/19957063 http://dx.doi.org/10.1007/s12575-009-9020-1 |
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author | Maioli, Emanuela Torricelli, Claudia Fortino, Vittoria Carlucci, Filippo Tommassini, Valentina Pacini, Adriana |
author_facet | Maioli, Emanuela Torricelli, Claudia Fortino, Vittoria Carlucci, Filippo Tommassini, Valentina Pacini, Adriana |
author_sort | Maioli, Emanuela |
collection | PubMed |
description | Rottlerin is a natural product isolated from Mallotus philippinensis. This polyphenolic compound, originally described as a selective inhibitor of PKCδ, can inhibit many other PKC-unrelated kinases and has a number of biological actions, including mitochondrial uncoupling effects. We recently found that Rottlerin inhibits the transcription factor nuclear factor κB in different cell types, causing downregulation of cyclin D1 and growth arrest. The present study was carried out to clarify the surprising lack of effect of Rottlerin on MCF-7 cell viability, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. We found that Rottlerin causes overestimation of the MTT test, leading to inconsistent results between cell number and cell viability. Rottlerin, however, strongly differs from other antioxidant polyphenols, which directly reduce tetrazolium salts, since it does not exhibit any reactivity toward the tetrazolium salts in vitro nor does it modulate lactate dehydrogenase activity. The interference in the MTT assay occurred only in cultured cells, concomitantly with a decrease in the energy charge. Because the same MTT overestimation was observed in the presence of uncoupling agents, we conclude that the Rottlerin artifact is linked to its uncoupling action that, by accelerating oxidative chain, accidentally results in enhanced MTT reduction. These results suggest caution in the use of the MTT assay in the presence of Rottlerin and uncouplers in general. |
format | Text |
id | pubmed-3056051 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-30560512011-03-13 Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers Maioli, Emanuela Torricelli, Claudia Fortino, Vittoria Carlucci, Filippo Tommassini, Valentina Pacini, Adriana Biol Proced Online Research Rottlerin is a natural product isolated from Mallotus philippinensis. This polyphenolic compound, originally described as a selective inhibitor of PKCδ, can inhibit many other PKC-unrelated kinases and has a number of biological actions, including mitochondrial uncoupling effects. We recently found that Rottlerin inhibits the transcription factor nuclear factor κB in different cell types, causing downregulation of cyclin D1 and growth arrest. The present study was carried out to clarify the surprising lack of effect of Rottlerin on MCF-7 cell viability, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. We found that Rottlerin causes overestimation of the MTT test, leading to inconsistent results between cell number and cell viability. Rottlerin, however, strongly differs from other antioxidant polyphenols, which directly reduce tetrazolium salts, since it does not exhibit any reactivity toward the tetrazolium salts in vitro nor does it modulate lactate dehydrogenase activity. The interference in the MTT assay occurred only in cultured cells, concomitantly with a decrease in the energy charge. Because the same MTT overestimation was observed in the presence of uncoupling agents, we conclude that the Rottlerin artifact is linked to its uncoupling action that, by accelerating oxidative chain, accidentally results in enhanced MTT reduction. These results suggest caution in the use of the MTT assay in the presence of Rottlerin and uncouplers in general. BioMed Central 2009-12-03 /pmc/articles/PMC3056051/ /pubmed/19957063 http://dx.doi.org/10.1007/s12575-009-9020-1 Text en Copyright ©2009 Maioli et al. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Maioli, Emanuela Torricelli, Claudia Fortino, Vittoria Carlucci, Filippo Tommassini, Valentina Pacini, Adriana Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers |
title | Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers |
title_full | Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers |
title_fullStr | Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers |
title_full_unstemmed | Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers |
title_short | Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers |
title_sort | critical appraisal of the mtt assay in the presence of rottlerin and uncouplers |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056051/ https://www.ncbi.nlm.nih.gov/pubmed/19957063 http://dx.doi.org/10.1007/s12575-009-9020-1 |
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