Abstract
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.
Highlights
Rottlerin, is a 5,7-dihydroxy2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)-8cinnamoyl-l,2- chromene, a pigmented plant product isolated from Mallotus philippinensis (Fig. 1)
Our laboratory showed for the first time that Rottlerin can prevent, independently from PKC, the activation of the transcription factor nuclear factor κB (NFκB), induced by either phorbol ester or oxidative stress in MCF-7 cells [5], HaCaT keratinocytes [6] and HT-29 cells, whose growth resulted to be arrested because of downregulation of cyclin D1, at both the protein and mRNA levels
In our previous paper [5], we found that MCF-7 cell viability was not altered by a 24-h Rottlerin treatment, a result that was in evident conflict with the inhibition of NFkB and cell proliferation, as evaluated by [3H]-thymidine incorporation into DNA
Summary
Been used as a PKCδ inhibitor [1] the selectivity of Rottlerin in inhibiting the PKCδ isoform has been recently questioned [2, 3] and ascribed to a likely indirect effect mediated by mitochondrial uncoupling and decrease in ATP content [4]. The followed procedure was the same as described above with some modifications: (a) The Rottlerin doses were 5, 20, 50, and 100 μM; (b) the incubation time with MTT was prolonged from 4 up to 24 h; (c) different media (DMEM, MEM, and RPMI 1640), enriched or not with 10% serum or 20μM NAD or NADH, were tested. In another set of experiments MCF-7 cells were treated for 30 min with equal doses (5 and 20 μM) of Rottlerin or the chemical uncoupler FCCP, before incubation with 10μl of MTT for 1 h at 37°C. The method permits to determine high-energy phosphates (ATP, ADP, and AMP) from which it is possible to calculate the energy charge of adenylates
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