Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers

Biological Procedures Online

Emanuela Maioli²  Claudia Torricelli²  Vittoria Fortino²  Filippo Carlucci¹  Valentina Tommassini¹  Adriana Pacini² 

[1] Department of Internal Medicine, Endocrine-Metabolic Science and Biochemistry, University of Siena, via Aldo Moro, 7-53100, Siena, Italy

[2] Department of Physiology, University of Siena, via Aldo Moro, 7-53100, Siena, Italy

关键词: mitochondrial uncoupling;    FCCP;    LDH;    MTT;    MCF-7 cell;    Rottlerin;   

Others  :  797145
DOI  :  10.1007/s12575-009-9020-1

 received in 2009-09-14, accepted in 2009-10-19,  发布年份 2009

【 摘 要 】

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.

【 授权许可】

   
2009 Maioli et al.

【 图 表 】

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