A proteasome-dependent inhibition of SIRT-1 by the resveratrol analogue 4,4'-dihydroxy- trans- stilbene.

Background and Aim: Resveratrol (RSV), is a stilbene-based compound exerting wide biological properties. Its analogue 4,4'-dihydroxy- trans -stilbene (DHS) has shown improved bioavailability and antiproliferative activity in vitro and in vivo . One of the hypotheses on how resveratrol works is based on SIRT1 activation. Since their strict structural similarities, we have explored a potential interaction between DHS and SIRT1, in comparison with the parental molecule.
Experimental Procedure: Timing of incubation and concentrations of DHS have been determined using MTT assay in normal human lung fibroblasts. Untreated, DHS- or RSV-treated cells were harvested and analysed by Western Blotting or RT-PCR, in order to evaluate SIRT1 levels/activity and expression, and by Cellular Thermal shift assay (CETSA) to check potential DHS or RSV-SIRT1 interaction. Transfection experiments have been performed with two SIRT1 mutants, based on the potential binding pockets identified by Molecular Docking analysis.
Results and Conclusion: We unexpectedly found that DHS, but not RSV, exerted a time-dependent inhibitory effect on both SIRT1 protein levels and activity, the latter measured as p53 acetylation. At the mRNA level no significant changes were observed, whereas a proteasome-dependent mechanism was highlighted for the reduction of SIRT1 levels by DHS in experiments performed with the proteasome inhibitor MG132. Bioinformatics analysis suggested a higher affinity of RSV in binding all SIRT1 complexes compared to DHS, except comparable results for complex SIRT1-p53. Nevertheless, both CETSA and SIRT1 mutants transfected in cells did not confirm this interaction. In conclusion, DHS reduces SIRT1 protein level, thereby inhibiting its activity through a proteasome-mediated mechanism.
Competing Interests: The authors declare no conflict of interest.
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