Rottlerin is a natural product isolated from Mallotus philippinensis. This polyphenolic compound, originally described as a selective inhibitor of PKCd, 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 kappa B 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.
Maioli, E., Torricelli, C., Fortino, V., Carlucci, F., Tommassini, V., Pacini, A. (2009). Critical Appraisal of the MTT Assay in the Presenceof Rottlerin and Uncouplers. BIOLOGICAL PROCEDURES ONLINE, 11(1), 227-240 [10.1007/s12575-009-9020-1].
Critical Appraisal of the MTT Assay in the Presenceof Rottlerin and Uncouplers
Maioli E.;Torricelli C.;
2009-01-01
Abstract
Rottlerin is a natural product isolated from Mallotus philippinensis. This polyphenolic compound, originally described as a selective inhibitor of PKCd, 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 kappa B 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.File | Dimensione | Formato | |
---|---|---|---|
1480-9222-11-1-9020.pdf
non disponibili
Tipologia:
Abstract
Licenza:
NON PUBBLICO - Accesso privato/ristretto
Dimensione
191.3 kB
Formato
Adobe PDF
|
191.3 kB | Adobe PDF | Visualizza/Apri Richiedi una copia |
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
https://hdl.handle.net/11365/25048
Attenzione
Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo