Aim: 

Rottlerin, a polyphenolic product isolated from Mallotus Philippinensis, has been widely used as a selective inhibitor of PKC delta, although it can inhibit many other kinases and has a number of biological effects. We recently found that Rottlerin inhibits the transcription factor NFkappaB in different cells, causing growth arrest via Cyclin D1 downregulation. This study is aimed to clarify the lack of Rottlerin effect on MCF-7 cell viability, assessed by the MTT test.

Methods: 

Total MCF-7 cell number was evaluate by using a Bürker chamber and trypan blue. MTT assay was performed in the presence or absence of cells. LDH assay was determined in culture medium in the presence or absence of Rottlerin. The cellular energy charge was evaluated by capillary zone electrophoresis.

Results: 

Rottlerin causes overestimation of the MTT test, resulting in discordant findings between cell number and cell viability. Rottlerin, however, strongly differs from other antioxidant polyphenols, which directly reduce tetrazolium salts even in the absence of cells, in that it does not exhibit any reactivity towards the salts in vitro nor it modulates LDH activity. The Rottlerin interference in the MTT assay occurs only in cultured cells, concomitantly with a decrease in the energy charge. Conclusion:

The observed MTT artifact is likely linked to the known Rottlerin mitochondrial uncoupling effect that, by accelerating oxidative chain, accidentally results in enhanced MTT reduction. We suggest caution in the use of the MTT assay as a proliferation/viability/toxicity test in the presence of Rottlerin.