Epigallocatechin-3-gallate inhibit the protein arginine methyltransferase 5 and Enhancer of Zeste homolog 2 in breast cancer bothin vitroandin vivo

Abstract

Histone methyltransferases are selectively catalyzing the methylation of lysine or arginine residues of target histone and non-histone proteins, classified as lysine methyltransferases and arginine methyltransferases. The EZH2 and PRMT5 catalyze trimethylation of H3 at K27 and symmetric dimethylation of H4 at R3 respectively. These histone repressive marks have been considered as hallmarks in cancer. Both PRMT5 and EZH2 over expressed in several cancers and have been considered as important target of drug development. As a result, many synthetic molecules as inhibitors of both PRMT5 and EZH2 are at different level of preclinical and clinical phases. Cancer atlas data analysis revealed that both PRMT5 and EZH2 had shown more than 90% amplification in breast cancer alone. We screened an array of phytocompounds towards the inhibition of PRMT5 and EZH2 usingin silico, in vitroassays. Among them Epigallocatechin-3-gallate (EGCG) has interacted with human PRMT5: MEP50 and EZH2 efficiently. The EGCG interacted within the SAM binding site, with a π-cation interaction at Lys 333 and H-bonds with Tyr324, Tyr334, Gly365, Leu437, and Glu444. Surface plasmon resonance analysis revealed that EGCG has strong binding affinity in nanomolar concentrations with both PRMT5-MEP50 than EZH2. Further in vitro methylation and cell-based assays proved the inhibitory potential of EGCG by reducing the catalytic products of PRMT5 and EZH2 i.e., H4R3me2s & H3K27me3 respectively and showed that it induced autophagy and apoptosis. Furthermorein vivo, mouse xenografts studies demonstrated that oral dosage, reduced tumor size significantly with reduction in proliferation marker Ki67 and these histone repressive marks. Finally conclude that inhibition of PRMT5 and EZH2 by EGCG potentially can be used to develop combined therapeutic approaches.

Ellagic acid: a potential inhibitor of enhancer of zeste homolog-2 and protein arginine methyltransferase-5

“…Surface plasmon resonance (SPR) analysis was conducted using the Biacore-T200 system (GE Healthcare) as described previously (33). Recombinant EZH2 and PRMT5-MEP50 complex (each 50µg) were diluted to 1mg/mL in 1x PBS pH 7.4 and immobilized onto a CM5 sensor chip using standard primary amine coupling.…”

“…To study the anti-proliferative properties of EA, the viability assay was conducted as described previously (33, 34) 5000 cells were grown per well in triplicate on 96-well plates and incubated overnight. The media was then replaced with DMEM only.…”

“…The trypan blue dye exclusion assay as previously described (33, 34) was employed in this study to assess cell viability following EA treatment. This assay is based on the principle that viable cells possess intact cell membranes that exclude the dye, while non-viable cells with compromised membranes take up the dye, rendering them visible under a light microscope.…”

“…The EB/AO double staining assay, as previously outlined (33, 34) was employed in this study to evaluate cell viability. This assay utilizes two fluorescent dyes: Acridine orange (AO) and ethidium bromide (EB).…”

“…The colony formation assay, based on established protocols (33, 34) with minor adjustments, was conducted to assess the impact of EA, on MDA-MB-231 cells. In this assay, 500 cells per well were seeded into 30mm culture dishes and allowed to adhere.…”