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The plant alkaloid chelerythrine binds to chromatin, alters H3K9Ac and modulates global gene expression

ABSTRACT: Chelerythrine (CHL), a plant alkaloid, possesses antimicrobial, anti-inflammatory and antitumor properties. Although CHL influences several key signal transduction pathways, its ability to interact directly with nucleoprotein complex chromatin, in eukaryotic cells has so far not been looked into. Here we have demonstrated its association with hierarchically assembled chromatin components viz. long chromatin, chromatosome, nucleosome, chromosomal DNA and histone H3 and the consequent effect on chromatin structure. CHL was found to repress acetylation at H3K9. It is more target-specific in terms of gene expression alteration and less cytotoxic compared to its structural analogue sanguinarine.

ORGANISM(S): Homo sapiens

PROVIDER: GSE80752 | GEO | 2017/04/28

SECONDARY ACCESSION(S): PRJNA319930

REPOSITORIES: GEO

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Project description:Sanguinarine (13-methyl[1,3]benzodioxolo[5,6-c]-1.3-dioxolo[4,5-i]phenanthridinium) is derived from the root of Sanguinaria canadensis L. (blood root), Chelidonium majus L. (Greater Celandine) and other poppy-fumaria species. Sanguinarine belongs to the benzophenanthridine alkaloid group, which are biosynthesized from phenylalanine in plants of the papaveraceae. This alkaloid has been reported to display a wide spectrum of biological effects, such as antifungal, antimicrobial, analgesic and moreover, anti-cancer properties via the induction of cell death. Utilizing dose-response studies with sanguinarine, we have shown that at two different concentrations sanguinarine induced over 95% apoptosis and oncosis respectively, in several cancer cell lines Also, the fact that oncosis and apoptosis are both induced in the same cell line under similar conditions and time points (with the sole exception being the concentration used) makes it a highly attractive model, because it facilitates easy and accurate comparison of both forms of cell death. These qualities make sanguinarine an ideal tool to study and compare oncosis with apoptosis. We are not aware of any other cell model system used to study oncosis that has all the above advantages.The objective of this study was to provide insight into the pathogenetic mechanisms of oncosis and apoptosis in cardiac myocytes in ischemic/reperfusion injury so that appropriate interventions could be designed to minimize injury and preserve myocardial function. We have shown here that primary cardiomyocytes, when exposed to sanguinarine at concentrations of 1.5 µg/ml (low dose) and 25 µg/ml (high dose) for 1 hour, displayed the classic biphasic pattern of apoptosis and oncosis. Using Illumina whole-genome expression microarrays, we have identified over 488 probes that were differentially expressed at p < 0.001 in cardiac myocytes between the low dose and high dose of sanguinarine after 1 hour of treatment; 78 of these changed more than 2-fold. Thus far, oncosis/necrosis has been thought of as uncontrolled and accidental, and lacking the features of apoptosis and autophagy. However, our findings and that of others reported in the literature indicate that this occurrence is not accidental but a tightly regulated physiological process. To our knowledge, this is the first report to present a model for the comparative study of apoptosis vs. oncosis in primary cardiomyocytes.

Project description:DNA binding anticancer agents cause alteration in chromatin structure and dynamics. Here, we report the dynamic interaction of the DNA intercalator and potential anticancer, plant alkaloid, Sanguinarine (SGR) with chromatin. Association of SGR with different levels of chromatin structure was found to be enthalpydriven. Apart from DNA it binds with comparable affinity to histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although, it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation dependent chromatin transcription. SGR mediated repression of epigenetic marks and the alteration of chromatin geography, also result in modulation of global gene expression. These data conclusively, show the anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context. The total RNA was isolated from control and treated cells using TRIZOL (Invitrogen) method. The Micromax direct labeling kit, MPS502 (PerkinElmer) was used to synthesize the labeled cDNA from 70 ug of total RNA and further process the hybridized cDNA on the array. All the steps were carried out according to manufacturerâs instructions (www.perkinelmer.com/lifesciences). The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Two arrays were used with at least one biological treatment of cells and dye swap experiment were included in the final analysis. . The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from both replicates.

Project description:DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. Here, we report the dynamic interaction of the DNA intercalator and potential anticancer, plant alkaloid, sanguinarine (SGR) with chromatin. Association of SGR with different levels of chromatin structure was found to be enthalpy driven. Apart from DNA, it binds with comparable affinity to histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR mediated repression of epigenetic marks and the alteration of chromatin geography also result in modulation of global gene expression. These data conclusively show that the anticancer DNA-binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context. The total RNA was isolated from control and treated cells using the TRIzol (Invitrogen) method. The Micromax direct labeling kit, MPS502 (PerkinElmer), was used to synthesize the labeled cDNA from 70 ug of total RNA and further process the hybridized cDNA on the array. All the steps were carried out according to manufacturer’s instructions (www.perkinelmer.com/lifesciences). The array slides were scanned immediately by the PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Five arrays were used with at least two biological treatments of cells, and dye-swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the downregulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from all five replicates.

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The plant alkaloid chelerythrine binds to chromatin, alters H3K9Ac and modulates global gene expression

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