Abstract
IntroductionCancer preventive effects of natural DNA-binding compounds (Curcumin, Quercetin, Resveratrol, Genistein, Fisetin, Coumarin, Apigenin, EGCG, Thymoquinon and Naringenin) involve activation of p53-mediated apoptosis and inhibition of NF-kappaB, PI3K, Wnt signalling pathways. However, molecular mechanism of this influence is not understood. We propose that it may be caused by interference of natural DNA-binding compounds with the normal modes of DNA packaging process.Material and methodsTo study chromatin destabilising effects of the compounds we used micrococcal nuclease digestion assay and live cell fluorescent imaging. In our experiment nuclei of HeLa cells were treated with DNA-binding compounds and then digested by micrococcal nuclease (NEB). DNA packaging in nucleosomes prevents DNA digestion, and loss of ordered nucleosome-protected ladder of DNA fragments in electrophoregrams may be observed only for compounds disabling chromatin. Curaxin CBL137, which is known to cause strong chromatin destabilisation, was taken as a positive control. DNA extracted from the nuclei of cells non-treated by the compounds after digestion with nuclease was used as a negative control.Results and discussionsIn Curaxin-treated samples, active DNA cleavage with nuclease was observed without the formation of bands corresponding to uncleaved fragments of DNA packed on nucleosomes. This assay did not reveal any effect of the natural DNA-binding compounds on chromatin structure as well as the study of H2B translocation by live cell fluorescent microscopy. It demonstrates that DNA-binding phytonutrients lack a strong destabilising effect on the histone core. Whilst, when histone H1 was studied, live cell imaging by fluorescent microscopy revealed a significant difference between cells treated with DNA-binding compounds and the control group cells. In particular, there was an increase in the proportion of cells with H1 localised in nucleoli or acquired intermediate localization. Effects of Genistein, Resveratrol, Curcumin, Fisetin increased during 24 hours, and Quercetin, Coumarin, EGCG, Thymoquinon and Naringenin influenced on histone H1 localization faster demonstrating maximum effects till 1 or 6 hour.ConclusionThus, weak chromatin-destabilising effects were shown for a number of natural DNA-binding compounds, although dynamics of their influence may vary. After cell treatment with natural DNA-binding compounds nucleosome core remains stable but histone H1 localization is influenced.This study was supported by RSF. 17-15-01526.
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