Effect of 5′‐aza‐2′‐deoxycytidine in comparison with and in combination to trichostatin a on dnmt1 and estrogen receptor alpha gene expression, cell growth inhibition, and apoptosis induction

Najmeh Sadeghi,1,* Fraidoon kavoosi,2

1. Department of physiology, faculty of medicine, Jahrom University of Medical Sciences, Jahrom, Iran
2. . Research center for non-communicable diseases, Jahrom University of medical sciences, Jahrom, Iran

Abstract

Introduction

Epigenetic regulation of gene expression is a reversible process, the term ‘epigenetics’ is the study of alterations caused by modifications, which occur during cell proliferation without any change in chromatin structure. these alterations include histone modifications, dna methylation, and rna interference (rnai) regulations. in human cells, dna methylation occurs on cytosines by dna methyltransferases (dnmts). dnmt activity induces dna hypermethylation resulting in chromatin compaction and gene silencing [1]. three dnmt enzymes have been identified, dnmt1, dnmt3a, and dnmt3b. dnmt3a and dnmt3b are responsible for establishing de novo methylation patterns, which are then maintained by dnmt1 [2]. in addition to dna methylation, histone acetylation plays a major role in chromatin structure and gene transcription, this process is often associated with a more ‘open’ chromatin conformation and gene reactivation. acetylation of histone is regulated by the competing activities of two enzymatic families including the histone acetyltransferases (hacs) and the histone deacetylases (hdacs) [3]. hdacs activities result in histone deacetylation and chromatin compaction. acetylation of histone achieved by hacs which decreases the affinity of the histone to dnd strand resulting in an open chromatin structure and gene expression. dna demethylating agents can inhibit dnmts by which inhibit dna hypermethylation leads to reactivation of gene expression [4]. furthermore, histone deacetylase inhibitors can inhibit hdacs activity and reactivate gene expression. previously we reported that dna demethylating agent genistein (ge) and histone deacetylase inhibitors trichostatin a (tsa) can inhibit dnmt1 activity and reactivate estrogen receptor alpha (erα) in hepatocellular carcinoma (hcc) which stimulated us to design the current article. the aim of this study was to evaluate the effect of dnmt inhibitor, 5′‐aza‐2′‐deoxycytidine (5‐azac) in comparison with and in combination to trichostatin a (tsa) on down-regulation of dnmt1, up-regulation of erα, cell growth inhibition, and apoptosis induction in human hcc plc/prf/5 cell lin

Methods

: human hcc plc/prf/5 cell were purchased from the national cell bank of iran‑pasteur institute and treated with tsa (1, 2.5, 5, 10, and 25 μm) and 5‐azac (1, 2.5, 5, 10 and 25 µm) except control groups to determine cell viability and ic50 assy. after 24 and 48 h, the cell viability was measured using mtt assay. to determine whether tsa and 5‐azac could down-regulate dnmt1 and up-regulate erα, real-time quantitative reverse transcription polymerase (qrt-pcr) was performed. in this regard, the cells were cultured and treated with tsa (2.5 μm) and 5‐azac (2.5), based on ic5o values, for 24 and 48. after treatment, the expression of the genes was obtained by qrt-pcr. finally, the apoptosis was determined by flow cytometry assy

Results

Both agents indicated a significant inhibitory effects on plc/prf/5 cell growth at different time periods (p <0.004). the 5‐azac indicated significant effect on down-regulation of dnmt1 and tsa up-regulated erα significantly. the effect of tsa in combination to 5‐azac on apoptosis was stronger than each agent alone (p<0.001).

Conclusion

: our result demonstrated that 5‐azac and tsa can down-regulate dnmt1 and up-regulate erα respectively. the modification of the gene expression resulted in significant cell growth inhibition and apoptosis induction.

Keywords

Trichostatin a, 5′‐aza‐2′‐deoxycytidine, tumor suppressor genes, hepatocellular carcinoma