Effects of trichostatin a on epigenetic reactivation of p14arf, p15ink4b, and p16ink4a genes in breast cancer
1. Department of Biology, faculty of science, University of Isfahan, Isfahan, I.R. Iran
Objective and aim: epigenetic modification of chromatin plays an important role in the regulation of gene transcription and expression. the modifications include acetylation, phosphorylation, methylation, sumoylation, ubiquitylation, and ribosylation, all of which can affect the gene expression significantly. post-translational modifications of histone tails provide an open chromatin structure resulting in activation of gene expression, the histones should be in an unlocked configuration to be in a permissible state for gene transcription. a balance between the activity of two group of enzymes including histone deacetylase (hdacs) and histone acetyltransferase (hac) determine the acetylation level of histone. histone deacetylase activity induces histone deacetylation leads to chromatin compaction and silenced gene. histone deacetylation of the promoter region of tumor suppressor genes leads to carcinogenesis. histone deacetylase inhibitors (hdacis) promote acetylation of histone which neutralizes the positive charge of the histone tails resulting in decreased affinity of the histone for the negatively charged dna strand. this change loosens the chromatin structure to an open configuration which enables the transcriptional machinery to access the dna chain leads to enhance gene transcription. according to chemical structure, hdacis can be classified into four classes including aliphatic acids, hydroxamates, cyclic peptides, and benzamides. previously, we evaluated the effects of genistein in comparison with 5-aza-2′-deoxycytidine on epigenetic reactivation of p14arf, p15ink4b, and p16ink4a genes in breast cancer. the results of the previous work encouraged us to design this study. the aim of the current study was to access the effect of trichostatin a (tsa) on epigenetic reactivation of p14arf, p15ink4b, and p16ink4a genes in breast cancer mda-mb-361 cell line.
Materials and methods: human breast cancer mda-mb-361 cell were purchased from the national cell bank of iran‑pasteur institute and cultured/treated with tsa with different concentrations of tsa (1, 5, 10, 15, and 20 μm) except control groups which incubated with dmso only. after 24 and 48 h, the cell viability was measured using mtt assay. to determine whether tsa could reactivate the gene expression, real-time quantitative reverse transcription polymerase (qrt-pcr) was performed. in this regard, the cells were cultured and treated with tsa (5 μm, based on ic5o values) for 24, 48. after treatment, relative expression of the genes was obtained by qrt-pcr.
Results: tsa indicated significant inhibitory effects and also activated p14arf, p15ink4b, and p16ink4a gene expression significantly. inhibitory effect of tsa was dose and time-dependent manner. the relative expression of these genes was 1.6 to 1.8 (p <0.003), 1.9 to 2.2 (p<0.001) and 1.7 to 2.1 (p<0.001) at 24 and 48 h respectively.
Conclusion: our result demonstrated that tsa can reactivate p14arf, p15ink4b, and
p16ink4a gene expression resulting in cell growth inhibition in human breast cancer mda-mb-361 cell line.
Keywords: trichostatin a, tumor suppressor genes, breast cancer