Kar Swayamsiddha, Sengupta Dipta, Deb Moonmoon, Shilpi Arunima, Parbin Sabnam, Rath Sandip Kumar, Pradhan Nibedita, Rakshit Madhumita, Patra Samir Kumar
Epigenetics and Cancer Research Laboratory, Biochemistry and Molecular Biology Group, Department of Life Science, National Institute of Technology, Rourkela, Odisha 769008, India.
Clin Epigenetics. 2014 Oct 13;6(1):20. doi: 10.1186/1868-7083-6-20. eCollection 2014.
DNA methylation mediates gene silencing primarily by inducing repressive chromatin architecture via a common theme of interaction involving methyl-CpG binding (MBD) proteins, histone modifying enzymes and chromatin remodelling complexes. Hence, targeted inhibition of MBD protein function is now considered a potential therapeutic alternative for thwarting DNA hypermethylation prompted neoplastic progress. We have analyzed the gene and protein expression level of the principal factors responsible for gene silencing, that is, DNMT and MBD proteins in MCF-7 and MDA-MB-231 breast cancer cell lines after treatment with various epigenetic drugs.
Our study reveals that the epigenetic modulators affect the expression levels at both transcript and protein levels as well as encourage growth arrest and apoptosis in MCF-7 and MDA-MB-231 cells. AZA, TSA, SFN, and SAM inhibit cell growth in MCF-7 and MDA-MB-231 cell lines in a dose-dependent manner, that is, with increasing concentrations of drugs the cell viability gradually decreases. All the epigenetic modulators promote apoptotic cell death, as is evident form increased chromatin condensation which is a distinct characteristic of apoptotic cells. From FACS analysis, it is also clear that these drugs induce G2-M arrest and apoptosis in breast cancer cells. Further, transcript and protein level expression of MBDs and DNMTs is also affected - after treatment with epigenetic drugs; the level of transcripts/mRNA of MBDs and DNMTs has consistently increased in general. The increase in level of gene expression is substantiated at the protein level also where treated cells show higher expression of DNMT1, DNMT3A, DNMT3B, and MBD proteins in comparison to untreated cells. In case of tissue samples, the expression of different DNMTs is tissue stage-specific. DNMT1 exhibits significantly higher expression in the metastatic stage, whereas, DNMT3A and DNMT3B have higher expression in the primary stage in comparison to the metastatic samples.
The epigenetic modulators AZA, TSA, SFN, and SAM may provide opportunities for cancer prevention by regulating the components of epigenetic gene-silencing machinery especially DNMTs and MBDs.
DNA甲基化主要通过涉及甲基化CpG结合（MBD）蛋白、组蛋白修饰酶和染色质重塑复合物的共同相互作用主题诱导抑制性染色质结构，从而介导基因沉默。因此，靶向抑制MBD蛋白功能现在被认为是阻止DNA高甲基化引发肿瘤进展的一种潜在治疗选择。我们分析了用各种表观遗传药物处理后，MCF-7和MDA-MB-231乳腺癌细胞系中负责基因沉默的主要因子，即DNMT和MBD蛋白的基因和蛋白表达水平。
我们的研究表明，表观遗传调节剂在转录和蛋白水平上影响表达水平，并促进MCF-7和MDA-MB-231细胞的生长停滞和凋亡。AZA、TSA、SFN和SAM以剂量依赖的方式抑制MCF-7和MDA-MB-231细胞系中的细胞生长，即随着药物浓度的增加，细胞活力逐渐降低。所有表观遗传调节剂都促进凋亡细胞死亡，这从染色质浓缩增加可以明显看出，染色质浓缩是凋亡细胞的一个明显特征。从流式细胞术分析也可以清楚地看出，这些药物在乳腺癌细胞中诱导G2-M期停滞和凋亡。此外，表观遗传药物处理后，MBD和DNMT的转录和蛋白水平表达也受到影响；一般来说，MBD和DNMT的转录本/mRNA水平持续增加。在蛋白水平上也证实了基因表达水平的增加，与未处理细胞相比，处理后的细胞显示出更高的DNMT1、DNMT3A、DNMT3B和MBD蛋白表达。在组织样本中，不同DNMT的表达具有组织阶段特异性。与转移样本相比，DNMT1在转移阶段的表达明显更高，而DNMT3A和DNMT3B在原发阶段的表达更高。
表观遗传调节剂AZA、TSA、SFN和SAM可能通过调节表观遗传基因沉默机制的组成部分，特别是DNMT和MBD，为癌症预防提供机会。
