Carotenoids are plant pigments abundant in fruits and vegetables. Carotenoids have been suggested to have a cancer preventive effect, However, the molecular mechanism of this action is not fully elucidated. Inhibition of the NFkB transcription system was suggested as a mechanism of cancer prevention by dietary electrophiles due to the interaction with critical cysteine residues in major players of the NFkB pathway, e.g. the key kinase IKK and the NFkB subunits. Carotenoids were shown to inhibit NFkB activity, however, intact carotenoids lack electrophylic groups and we have previously demonstrated that carotenoid derivatives but not the intact carotenoids activate the Nrf2 transcription system. Thus, our aim was to determine whether carotenoid derivatives inhibit NFkB and the molecular mechanism of this inhibition.
We analyzed the structure-activity relationship of a series of dialdehyde carotenoid derivatives in NFkB inhibition. These compounds inhibited NFkB-driven reporter gene expression as well as several stages of the pathway in T47D mammary cancer cells. Moreover, the activity of the carotenoid derivatives depended on the reactivity of the electrophylic group in reactions such as Michael addition to SH groups in proteins. Specifically, carotenoid derivatives directly affected the NFkB machinery at two stages: the key regulatory enzyme IKK, as well as the p65 subunit of the transcription factor. Direct interaction with IKKβ was found inacellularin vitro kinase assay with a recombinant enzyme. Inhibition of p65 transcriptional activity was found in a reporter gene assay conducted in the presence of excess p65. A reducing agent partly reversed this effect. Inhibition of p65 activity at least partially results from reduced binding to DNA as evident from EMSA experiments. Interestingly, the binding of a mutant p65 where cysteine 38 was converted to serine was not affected by the derivative, proving that the interaction with this specific cysteine is crucial for the inhibition by carotenoid derivatives. The same trend was observed with a cysteine-mutant of IKK.
In conclusion, we suggest that electrophylic carotenoid derivatives contribute to cancer prevention by inhibition of the NFkB transcription system. Strategic thiol groups of both IKK and p65 play a pivotal role in this process.
