Department of Medical Biochemistry, Faculty of Medicine in Hradec Kralove, Charles University, Hradec Kralove, Czech Republic.
Institute for Clinical Biochemistry and Diagnostics, Faculty of Medicine in Hradec Kralove, Charles University and University Hospital Hradec Kralove, Hradec Kralove, Czech Republic.
PLoS One. 2019 Jan 17;14(1):e0210889. doi: 10.1371/journal.pone.0210889. eCollection 2019.
Histone modifications play a key role in the epigenetic regulation of gene transcription in cancer cells. Histone acetylations are regulated by two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs are increased in ovarian carcinomas and they are involved in carcinogenesis and resistance to chemotherapeutic agents. In our study we investigated anticancer effect of HDAC inhibitor sodium butyrate (NaBu) on cisplatin-sensitive and cisplatin-resistant ovarian cell lines A2780 and A2780cis. A2780 and A2780cis were treated with NaBu alone or in combination with cisplatin (CP). NaBu inhibited the growth of both cell lines and enhanced cytotoxic effect of CP. Exposure to NaBu for 24 h induced cell cycle arrest. The expressions of EMT-related genes and proteins were further investigated by qPCR and western blot analysis. Loss of E-cadherin has been shown to be crucial in ovarian cancer development. We found that NaBu dramatically induce expression of E-cadherin gene (CDH1) and protein levels in A2780 and A2780cis. We investigated correlation between transcription and methylation of CDH1gene. Methylation level analysis in 32 CpG sites in CDH1 gene (promoter/exon1 regions) was performed using bisulfite NGS (Next Generation Sequencing). We found that cisplatin-resistant cell line A2780cis cells differ from their cisplatin-sensitive counterparts in the CDH1 methylation. Methylation in A2780cis cells is elevated compared to A2780. However, NaBu-induced expression of CDH1 was not accompanied by CDH1 demethylation. NaBu treatment induced changes in expression of EMT-related genes and proteins. Interestingly E-cadherin zinc finger transcriptional repressor SNAIL1 was upregulated in both cell lines. Mesenchymal marker vimentin was downregulated. Matrix metalloproteases (MMPs) are necessary for pericellular proteolysis and facilitate migration and invasion of tumour cells. NaBu induced mRNA expression of MMPs, mild changes in activities of gelatinases MMP2 and MMP9 were detected. Our data demonstrate that NaBu sensitizes cisplatin-resistant ovarian cancer cells, re-established E-cadherin expression, but it was not able to reverse the EMT phenotype completely.
组蛋白修饰在肿瘤细胞中的基因转录表观遗传调控中起着关键作用。组蛋白乙酰化受两类酶调控，即组蛋白乙酰转移酶（HATs）和组蛋白去乙酰化酶（HDACs）。卵巢癌中 HDACs 增加，它们参与致癌作用和对化疗药物的耐药性。在我们的研究中，我们研究了 HDAC 抑制剂丁酸钠（NaBu）对顺铂敏感和耐药的卵巢癌细胞系 A2780 和 A2780cis 的抗癌作用。A2780 和 A2780cis 单独用 NaBu 或与顺铂（CP）联合处理。NaBu 抑制了这两种细胞系的生长，并增强了 CP 的细胞毒性作用。暴露于 NaBu 24 小时诱导细胞周期停滞。通过 qPCR 和 Western blot 分析进一步研究 EMT 相关基因和蛋白的表达。已经表明，E-钙粘蛋白的丢失在卵巢癌的发展中是至关重要的。我们发现 NaBu 可显著诱导 A2780 和 A2780cis 中 E-钙粘蛋白基因（CDH1）的表达和蛋白水平。我们研究了 CDH1 基因转录和甲基化之间的相关性。使用亚硫酸氢盐 NGS（下一代测序）对 CDH1 基因（启动子/外显子 1 区域）中的 32 个 CpG 位点进行了甲基化水平分析。我们发现，与顺铂敏感的 A2780 细胞相比，顺铂耐药的 A2780cis 细胞在 CDH1 甲基化上存在差异。与 A2780 相比，A2780cis 细胞的甲基化水平升高。然而，NaBu 诱导的 CDH1 表达并不伴随着 CDH1 去甲基化。NaBu 处理诱导 EMT 相关基因和蛋白表达的变化。有趣的是，两个细胞系中 E-钙粘蛋白锌指转录抑制因子 SNAIL1 上调。间充质标志物波形蛋白下调。基质金属蛋白酶（MMPs）是细胞外基质蛋白水解所必需的，促进肿瘤细胞的迁移和侵袭。NaBu 诱导 MMPs 的 mRNA 表达，检测到明胶酶 MMP2 和 MMP9 的活性有轻微变化。我们的数据表明，NaBu 使顺铂耐药的卵巢癌细胞敏感，重新建立了 E-钙粘蛋白的表达，但不能完全逆转 EMT 表型。
