RESUMO
BACKGROUND: 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. Here, we describe a cell-based assay system for screening gene reactivation. METHODS: A cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system. RESULTS: Following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation. CONCLUSIONS: The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Mama/citologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteínas Luminescentes/metabolismo , Nitrorredutases/metabolismo , Antineoplásicos/farmacologia , Azacitidina/farmacologia , Proteínas de Transporte/genética , Células Cultivadas , Citomegalovirus/genética , Metilação de DNA , Decitabina , Epigênese Genética , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos , Humanos , Ácidos Hidroxâmicos/farmacologia , Proteínas Luminescentes/genética , Nitrorredutases/genética , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Lipossomas Unilamelares , Vorinostat , Proteína Vermelha FluorescenteRESUMO
ANKRD11 is a putative tumour suppressor gene in breast cancer, which has been shown in our laboratory to be a co-activator of p53. Our data suggest that down-regulation of ANKRD11 is associated with breast tumourigenesis. Breast cancer cell lines treated with DNA demethylating agents resulted in up-regulation of ANKRD11 expression suggesting that promoter DNA methylation may be responsible for its down-regulation. The transcriptional activity of a CpG-rich region 2kb upstream of the transcription initiation site of ANKRD11 was investigated using dual-luciferase reporter assays. The constructs carrying -661 to -571 bp promoter sequence showed significant transcriptional activity. Using the SEQUENOM Epityper Platform, the region between -770 and +399 bp was analysed in 25 breast tumours, four normal breast tissues and five normal blood samples. The region between -770 and -323 bp was shown to be frequently methylated in breast tumours. The methylation patterns of all analysed CpGs in this region were identical in the normal and tumour samples, except for a 19 bp region containing three CpG sites. These sites had significantly higher levels of methylation in tumours (40%) compared to normal samples (6%). Our findings support the role of ANKRD11 as a tumour suppressor gene and suggest that aberrant DNA methylation of three CpGs in a 19 bp region within the ANKRD11 promoter may be responsible for its down-regulation in breast cancer.
Assuntos
Neoplasias da Mama/genética , Metilação de DNA , Genes Supressores de Tumor , Proteínas Repressoras/genética , Sequência de Bases , Linhagem Celular Tumoral , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/genética , Feminino , Humanos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , DNA Metiltransferase 3BRESUMO
DNA methylation, which often occurs at the cytosine residue of cytosine-guanine dinucleotides, is critical for the control of gene expression and mitotic inheritance in eukaryotes. DNA methylation silences gene expression either by directly hindering the access of transcription factors to the target DNA, or through recruitment of histone deacetylases to remodel the chromatin structure to an inactive state. Aberrant hypermethylation of tumor suppressor genes is commonly associated with the development of cancer. A number of anti-cancer agents have been developed that function through demethylation, reversing regional hypermethylation to restore the expression of tumor suppressor genes. Azacitidine and decitabine are used in the clinic, but their applications are limited to myelodysplastic syndrome and other blood-related diseases. Despite the potency of these drugs, their broader clinical application is restricted by cytotoxicity, nonspecific targeting, structural instability, catabolism, and poor bioavailability. Further improvements in the delivery systems for these drugs could overcome the issues associated with inefficient bioavailability, whilst facilitating the administration of combinations of demethylating agents and histone deacetylase inhibitors to enhance efficacy. This review focuses on the current limitations of existing demethylating agents and highlights possible approaches using recent developments in drug delivery systems to improve the clinical potential of these drugs.