CriticalAppraisaloftheMTTAssayinthePresenceofRottlerin1

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.

Key Words: Rottlerin - MCF-7 cell - MTT - LDH - FCCP - mitochondrial uncoupling

Introduction

Rottlerin (also called mallotoxin or kamala), is a 5,7-dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)-8-cinnamoyl-l,2- chromene, a pigmented plant product isolated from Mallotus philippinensis (Fig. 1 ). Since 1994, Rottlerin has been used as a PKCδ inhibitor (1) although 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). 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induced by either phorbol ester or oxidative stress in MCF-7 cells (5), HaCaT keratinocytes (6) and HT-29 cells (unpublished results), whose growth resulted to be arrested because of downregulation of cyclin D1, at both the protein and mRNA levels. Although the molecular mechanism is not definitively clarified, the prevention of the NFκB activation process was likely achieved through both Rottlerin inhibition of protein kinases (7, 8) and Rottlerin free radicals scavenging activity (9). Indeed, NFκB can be activated by a number of pathways and is a redox-sensitive transcription factor for key molecules involved in inflammation, cancer progression, cell cycle control, and protection against apoptosis (10).

Fig. 1 Rottlerin structure.

However, 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. Since the measurement of cell viability was based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan crystal by mitochondrial dehydrogenases (11), in the current study we revisited our previous results, checking for a possible interference of Rottlerin in the MTT assay.