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. 2020 Nov;10(11):484.
doi: 10.1007/s13205-020-02473-1. Epub 2020 Oct 23.

Epigallocatechin gallate inhibits HeLa cells by modulation of epigenetics and signaling pathways

Madhumitha Kedhari Sundaram  1 , Shafiul Haque  2 , Pallavi Somvanshi  3 , Tulika Bhardwaj  3 , Arif Hussain  1
Affiliations

Epigallocatechin gallate inhibits HeLa cells by modulation of epigenetics and signaling pathways

Madhumitha Kedhari Sundaram et al. 3 Biotech. 2020 Nov.

Abstract

This study examines the effect of epigallocatechin gallate (EGCG) on signaling pathways, epigenetic modulators and tumour suppressor genes in cervical cancer cells, HeLa. qRT-PCR, ELISA-based enzymatic assays and in silico studies were used to catalogue the modulation of these genes by EGCG treatment. qRT-PCR showed transcriptional modulation of several epigenetic modifiers including DNA methyltransferases and histone modifiers (DNMT1, DNMT3B, DNMT3A, AURKA, AURKC, AURKB, KDM4A, KDM5C, PRMT7, PRMT6, UBE2B, HDAC5, HDAC6, HDAC7 and HDAC11. Furthermore, ELISA-based assays showed that EGCG lowered the activity of DNA methyltransferases, histone deacetylases and histone methyltransferases (H3K9). Molecular docking results suggests that EGCG may competitively inhibit some epigenetic enzymes (DNMT1, DNMT3A, HDAC2, HDAC3, HDAC4, HDAC7 and EZH2). A functional outcome of these epigenetic alterations could be inferred from the reversal of promoter hypermethylation of tumour suppressor genes by quantitative methylation array and transcriptional re-expression of tumour suppressor genes including TP73, PTEN, SOCS1, CDH1, RARβ, and DAPK1 by qRT-PCR. Downregulation of key signaling moieties of PI3K, Wnt and MAPK pathways, cell cycle regulators, metastasis regulators and pro-inflammatory moieties including TERT, CCNB1, CCNB2, MMP2, MMP7. PIK3C2B, PIK3CA, MAPK8 and IL6 was also observed. In silico protein-protein interaction network analysis followed by KEGG analysis discerned the active participation of gene sets towards cancer pathways. This study comprehensively explains EGCG's anti-cancer mechanism via the synchronized transcriptional alteration of several molecular targets across different signaling pathways and reversal of tumour suppressor gene silencing through modulation of epigenetic enzymes.

Keywords: Cancer; EGCG; Epigenetics; Signaling pathway; Tumour suppressor gene.

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Conflict of interest statement

Conflict of interestThe authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
a Effect of EGCG on various genes involved in carcinogenesis RQ plot of tumour suppressor genes and genes involved in cell cycle regulation and cell-signaling pathways in HeLa cells after treatment with 50 µM of EGCG for 48 h is shown. Fold change was calculated by ΔΔCT analysis in comparison to untreated HeLa cells after global normalization. Values are means ± SD of three independent experiments (p ≤ 0.05). b Effect of EGCG on genes involved in chromatin modification RQ plot of genes involved in the chromatin modification whose expression in HeLa cells is modulated following treatment with 50 µM EGCG for 48 h. Fold change was calculated by ΔΔCT analysis in comparison to untreated HeLa cells after global normalization. Values are means ± SD of three independent experiments (p ≤ 0.05)
Fig. 2
Fig. 2
Molecular docking analysis of sulforaphane with epigenetic enzymes and biochemical inhibition of enzyme activity EGCG was docked with the crystal structures of various enzymes using the docking server, Swiss Dock and analyzed using UCSF Chimaera. The substrate binding site of the enzymes was identified using literature resources and cavity predicting software. The substrate-binding cavity is shown in hydrophobic representation in cyan colour with the catalytic residue coloured green. The predicted interaction of EGCG (blue) with the active site residues (cyan) in the substrate binding cavity of a DNMT1, b DNMT3A, c HDAC2, d HDAC3, e HDAC4, f HDAC7 g EZH2 is shown. The bound results were then compared to known crystal structures of inhibitors for validation. h Effect of EGCG on activity of DNMT, HDAC and HMT-H3K9 in HeLa cells. Nuclear extract of HeLa cells was treated with 50 µM EGCG to observe the direct inhibition of enzymatic activity of DNMTs, HDACs and HMT-H3K9s using an ELISA-based assay. EGCG demonstrated significant inhibition of the activity of all enzyme families. Values are represented in comparison to untreated control and are means ± SD of three independent experiments. (*p ≤ 0.05)
Fig. 3
Fig. 3
Effect of EGCG on global DNA methylation in HeLa cells a The global methylation level of DNA in HeLa cells treated with 50 µM EGCG for 24 h and 48 h was assessed using an ELISA-based assay. DNA from untreated HeLa cells were used as control. EGCG significantly decreased the levels of global DNA methylation in HeLa cells in a time-dependent manner. The decrease is methylation level is represented as a percentage of the untreated control. Values are means ± SD of three independent experiments. (*p ≤ 0.05). b Effect of EGCG on gene specific DNA methylation in DNA extracted from HeLa cells treated with 50 µM EGCG for 48 h was subjected to methylation-dependent restriction digestion. The products of the digest were used as template for a PCR reaction using Human Tumor Suppressor Genes EpiTect Methyl II Signature PCR Array, to determine the 5´ CpG island promoter methylation level of tumour suppressor genes. EGCG significantly decreased the promoter methylation levels in HeLa cells in comparison to untreated control. Values are means ± SD of three independent experiments (*p ≤ 0.05)
Fig. 4
Fig. 4
a Protein–protein interaction network protein–protein interaction network of genes which are differentially modulated following EGCG treatment of HeLa cells is shown using k-means clustering. STRING Cytoscape App was used after scaling of gene expression data; evaluation of the clusters was performed by estimating an average silhouette width as nine. b KEGG pathway enrichment analysis KEGG pathway enrichment analysis of genes differentially modulated following EGCG treatment of HeLa cells predicts relationship between biological response variable and metabolic pathway based on linear model computing p-statistic score
Fig. 5
Fig. 5
Overview of EGCG mechanism of action EGCG was observed to bring about transcriptional modulation of several genes. A connection between the modulated genes has been represented in this figure to illustrate the mechanism behind the anti-carcinogenic effect of EGCG
See this image and copyright information in PMC

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