HTP Nutraceutical Screening for Histone Deacetylase Inhibitors and Effects of HDACis on Tumor-suppressing miRNAs by Trichostatin A and Grapeseed (Vitis vinifera) in HeLa cells.

Abstract

Aggressive tumor malignancies are a consequence of delayed diagnosis, epigenetic/phenotype changes and chemo-radiation resistance. Histone deacetylases (HDACs) are a major epigenetic regulator of transcriptional repression, which are highly overexpressed in advanced malignancy. While original chemotherapy drugs were modeled after phytochemicals elucidated by botanical screenings, HDAC inhibitors (HDACi) such as apicidin, trichostatin A (TSA) and butyrate were discovered as products of fungus and microbes, in particular, gut microbiota. Therefore, a persistent question remains as to the inherent existence of HDACis in raw undigested dietary plant material. In this study, we conduct a high-throughput (HTP) screening of ~1,600 non-fermented commonly used nutraceuticals (spices, herbs, teas, vegetables, fruits, seeds, rinds etc.) at (<600 μg/ml) and food-based polyphenolics (<240 μg/ml) for evidence of HDAC activity inhibition in nuclear HeLa cell lysates. Human HDAC kinetic validation was performed using a standard fluorometric activity assay, followed by an enzymatic-linked immuno-captured ELISA. Both methods were verified using HDACi panel drugs: TSA, apicidin, suberohydroxamic acid, M344, CL-994, valproic acid and sodium phenylbutyrate. The HTP screening was then conducted, followed by a study comparing biological effects of HDACis in HeLa cells, including analysis of whole-transcriptome non-coding RNAs using Affymetrix miRNA 4.1-panel arrays. The HTP screening results confirmed 44/1600 as potential HDACis to which 31 were further eliminated as false-positives. Methodological challenges/concerns are addressed regarding plant product false-positives that arise from the signal reduction of commercial lysine development reagents. Only 13 HDACis were found having an IC50 under <200 μg/ml: Grapeseed extract (Vitis vinifera), Great burnet root (Sanguisorba Officinalis), Babul (Acacia arabica), Chinese gallnut (Melaphis chinensis), Konaberry extract (Coffea arabica), Uva Ursi (Arctostaphylos uva ursi), Green tea (Camellia sinensis), Meadowsweet (Filipendula ulmaria), Sassafras (Sassafras officinale), Turkey rhubarb (Rheum palmatum), epigallocatechin gallate (EGCG), gossypol and gallic acid. Next, we investigate the biological consequence of HDACi panel drugs in HeLa cells, where the data suggest predominant effects are anti-mitotic rather than cytotoxic. Lastly, differential effects of TSA vs. GSE at sub-lethal concentrations tested on HeLa cells show 6,631 miRNAs expressed in resting cells, 35 significantly up-regulated (TSA) and 81 up-regulated (GSE), with several miRNAs overlapping in the upward direction by both GSE and TSA (e.g. hsa-miR-23b-5p, hsa-miR-27b-5p, hsa-miR-1180-3p, hsa-miR-6880-5p and hsa-mir-943). Using DIANA miRNA online tools, it was determined that GSE and TSA simultaneously cause overexpression of similar miRNAs predicted to destroy the following influential oncogenes: NFkB, NRAS, KRAS, HRAS, MYC, TGFBR1, E2F1, E2F2, BCL21, CDKN1A, CDK6, HIF1a, and VEGFA. The data from this study show that plant- based HDACis are relatively rare, and can elicit a similar pattern to TSA in up-regulating miRNAs involved with tumor suppression of HeLa cervical carcinoma.