Development of a novel cell-based assay system EPISSAY for screening epigenetic drugs and liposome formulated decitabine

DNA methylation and histone modification are the two major epigenetic mechanisms catalyzed by DNMTs and HDACs, respectively [1]. HDACs remove the acetyl groups from histones, whilst DNMTs catalyse the transfer of a methyl group from S-adenosylmethionine to the 5-carbon position of the cytosine pyrimidine ring, both leading to the condensation of chromatin to its inactive state [2, 3]. In cancer cells, an abundance of hypo-acetylated histones is usually associated with DNA hyper-methylation and gene silencing [4]. These findings are the basis for the development of HDAC and DNMT inhibitors as cancer therapeutics. Such compounds block the activity of HDACs and DNMTs, leading to increased expression of epigenetically silenced genes which mediate cellular and metabolic changes such as cell growth arrest, differentiation and apoptosis [5‐9].

Hydrophobic vorinostat (suberoylanilide hydroxamic acid, SAHA) and hydrophilic decitabine (5-aza-2^′-deoxycytidine, Dacogen) are US Food and Drug Administration (FDA) approved HDAC and DNMT inhibitors for the treatment of cutaneous T-cell lymphoma and myelodysplastic syndrome, respectively [10, 11]. The combination of vorinostat and decitabine have been shown to have promising activity in patients with myelodysplastic syndrome without significant toxicity in a phase I clinical trial [12]. Under neutral conditions, decitabine has a reported half-life of 7 days at 4°C or 21 hours at 37°C in vitro[13]. However, decitabine is degraded more rapidly in vivo with a half-life of only 25 minutes [13]. Such chemical instability of decitabine has led to its administration in the clinic as a cold and continuous intravenous infusion in an effort to reach the maximal-tolerated doses required to achieve clinical response [14, 15].

The development of drug formulation using nanotechnology (e.g. liposomes) has been used to improve drug stability [16, 17]. Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. A commonly used cell-based assay for both DNMT and HDAC inhibitors is the quantification of the re-expression of known epigenetically-silenced genes by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis [5, 18]. However, this traditional approach is not high-throughput and may produce gene-specific results. Other assays that have been used include estimation of global DNA methylation using capillary electrophoresis, DNA digestion with methylation-sensitive restriction enzymes, or analysis of specific DNA methylation using bisulfite sequencing and methylation-specific PCR [19]. However, these assay systems designated for assaying DNMT or HDAC inhibitors are time-consuming, cumbersome and subject to misinterpretation [20‐22]. Consequently, the rapid identification and validation of novel epigenetic drugs are hampered due to the lack of an efficient screening method.

In this study, a cell-based assay system was developed to compare the activity of different epigenetic drugs. This assay system is based on mammalian MCF10A cells expressing a fusion protein between red-fluorescent protein (RFP) and bacterial nitroreductase (TMnfsB) driven by CMV promoter. Epigenetic silencing has been shown to silence genes driven by CMV promoter in both stably transfected cells and transgenic pigs [23, 24]. Silenced CMV promoter driven genes were shown to be reactivated after treatment with epigenetic drugs such as butyrate, trichostatin A and decitabine [23]. Human cells expressing TMnfsB are able to metabolize the monofunctional alkylating prodrug CB1954 (5-(azaridin-1-yl)-2,4-dinitro-benzamide) to highly cytotoxic hydroxylamino- and amino-derivatives, which induce rapid cell death [25]. Therefore, TMnfsB was utilized as a tool to obtain clones with inactivated CMV promoters. The TMnfsB open reading frame has been codon optimized to increase the sensitivity of stable human cell lines to the prodrug CB1954 [26]. An assay system for gene reactivation was developed by identifying clones where expression of RFP-TMnfsB was suppressed at the transcriptional level, but could be re-established by subsequent treatment with epigenetic drugs. Since RFP expression in these clones is low, it was used as a signal to evaluate the reactivation of gene expression by flow cytometry. Using this newly developed assay system, it was shown that decitabine which encapsulated in the liposomes has a higher gene restoring ability than pure decitabine, zebularine and RG108.

EPISSAY, a cell-based assay system for screening of epigenetic drugs was developed based on the human non-malignant breast epithelial cell line MCF10A expressing the well-characterized CMV promoter driving RFP fused with a mammalianized version of the bacterial nitroreductase nfs gene. The nfs gene has been used in gene-directed enzyme prodrug therapy [37] since treatment of mammalian cells expressing nfs with CB1954 results in its chemical reduction to cytotoxic metabolites. Exposure of the derivative MCF10A with CB1954 was used as a strategy for the selection of cell lines with silenced nfs genes.

The EPISSAY was verified by treatment with the known epigenetic drugs decitabine, zebularine or vorinostat; all of which resulted in increased red-fluorescence due to reactivation of the CMV promoter. There was a linear relationship between nfs expression and the red-fluorescent signal confirming that levels of gene message and translated protein are directly related. The response was further confirmed by measuring expression levels of known independent endogenous genes TXNIP[5] and ANKRD11[34].

EPISSAY could be a time-saving assay for screening compounds with gene reactivating activity. Standard methodologies used to assess epigenetic compounds are based on quantitative real-time PCR and western blot analysis of genes known to be silenced in a particular cell line. For example, quantification of the re-expression of an endogenous gene p16 in human T24 bladder carcinoma cell line was previously used [38]. Such approaches are time-consuming as they require cell collection for RNA and protein extractions prior to analysis. Other cell-based assay systems which use exogenous expression of genes (e.g. Escherichia coli β-D-galactosidase gene with and green fluorescent reporter) have previously been investigated for their potential in screening epigenetic drugs by using fluorescent microscopy and plate readers. However, these other systems have limitations such as the non-quantitative data obtained and/or additional sample treatments required (e.g Paraformaldehyde fixing, the addition fluorogenic compounds) prior to screening [18‐20] (Additional file 4). EPISSAY requires limited sample preparation, may be formatted for multi-well plates, and rapid results can be generated from RFP reading using flow cytometry to obtain quantitative data.

Decitabine is a demethylating agent that is FDA approved as an anti-cancer agent [13]. Since decitabine is degraded in vivo with a half-life of only 25 minutes, daily treatments are required to maintain appropriate drug levels both in vitro and in vivo[39]. To improve the stability and bioavailability of decitabine, the drug was encapsulated in PEGylated liposomes, as liposomes are known to protect drugs from degradation and allow controlled release of drug into the environment [40]. This formulation achieved an encapsulation efficiency of ~50%. Only 3.3 mol% of PEG 2000 was used in this study as a higher PEG content is known to reduce adsorption of liposomes onto cells [41].

Liposomes were extruded through filters with defined pore size (200 nm and 400 nm) to obtain unilamellar liposomes. Although extrusion does not affect the encapsulation efficiency [42], it narrowed the size distribution of the liposomes from 1 μm to approximately 150 nm. The smaller size of the drug-loaded liposomes has been reported to passively targeting disease tissues due to their enhanced angiogenesis [43].

We used the EPISSAY system to determine if liposomal encapsulation enhanced the gene reactivating activity of decitabine. Following 72 hours of treatment, decitabine encapsulated in unilamellar liposomes showed 50% more potency than pure decitabine, suggesting that decitabine was protected in the liposomes and slowly released into the media. These results were supported by a controlled release study comparing the drug release of decitabine from unilamellar and multilamellar liposomes. This showed that the release rate of decitabine from unilamellar liposomes was slower, suggesting unilamellar liposomal formulation may decrease the rate of degradation of decitabine by providing protection to the drug. In addition, the liposomal formulation and the presence of phospholipids in the cell media could also contribute to the enhancement of decitabine activity [44, 45].

Collectively, the liposomal decitabine that was synthesised here was validated as a more potent epigenetic drug. However, we have only confirmed this in vitro. An in vivo study of liposomal decitabine is recommended to assess its applicability for clinical use, and to confirm if the present limitations of decitabine use in the clinic could be overcome by this formulation. The use of liposomes/PEG to encapsulate drugs to improve their bio-availability and stability is now gaining momentum with a number of drugs eg doxorubicin [17], rhenium radionuclides [46] and dexamethasone phosphate [47], liposome-encapsulated doxorubicin now having FDA approval.

In this pilot study, we have constructed and evaluated a novel bioassay for epigenetic compounds. The readout of the EPISSAY system is red-fluorescence, which may allow the adaptation of the assay system to a multi-well format allowing high throughput, rapid, and cheap bioassay in the future. EPISSAY was successful in providing evaluation of different liposomal formulations of decitabine. The EPISSAY can detect the gene reactivating effects of decitabine, zebularine or vorinostat. Linear correlation between the message of an endogenous gene ANKRD11 and red-fluorescent reading has been shown in the EPISSAY cells treated with pure decitabine and unilamellar liposomes-formulated decitabine (Additional file 5).

Using SEQUENOM MassARRAY EpiTYPER, no major changes in methylation of the CMV promoter was detected in the EPISSAY cells before and after treatments with decitabine (Additional file 6 and 7). Although vorinostat is known as a HDAC inhibitor to activate gene expression, zebularine and decitabine are usually considered to function as demethylating agents or DNMT inhibitors [48]. However, there are now multiple studies that show these agents can also function as HDAC inhibitors [49‐51]. This suggests that the TMnfsB gene was most likely silenced by histone modification rather than direct methylation of the CMV promoter. There is a potential of adopting this assay as a high throughput, rapid and low cost epigenetic drug screening platform are unique aspects of the EPISSAY system. We conclude that our EPISSAY bioassay system provides a novel and rapid system to screen the efficiencies of epigenetic and newly formulated drugs for gene reactivation.