Demethylation and alterations in the expression level of the cell cycle-related genes as possible mechanisms in arsenic trioxide-induced cell cycle arrest in human breast cancer cells
Demethylation and alterations in the expression level of the cell cycle-related genes as possible mechanisms in arsenic trioxide-induced cell cycle arrest in human breast cancer cells
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DOI：
10.1177/1010428317692255
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发表时间：
2017-02-01
期刊：
影响因子：
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通讯作者：
Ghaffari, Seyed H.
中科院分区：
文献类型：
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作者：
Moghaddaskho, Farima;Eyvani, Haniyeh;Ghaffari, Seyed H.
三氧化二砷（As 2 O3）已被临床用作抗肿瘤剂。其作用机制主要是诱导细胞凋亡和细胞周期阻滞。然而，其通过细胞周期阻滞的抗癌作用的详细分子机制知之甚少。此外，As 2 O3已被证明是一种潜在的DNA甲基化抑制剂，诱导DNA低甲基化。我们推测As 2 O3可能通过干扰DNA甲基化模式来影响细胞周期调控基因的表达。为了探索这一点，我们研究了乳腺癌细胞系和正常乳腺组织样本中的24个细胞周期基因的启动子甲基化状态的甲基化特异性聚合酶链反应和/或限制性内切酶为基础的方法。分别用实时荧光定量聚合酶链反应和流式细胞术分析基因表达水平和细胞周期分布。我们的甲基化分析表明，只有RBL 1（p107），RASSF 1A，和细胞周期蛋白D2的启动子在研究的乳腺癌细胞系异常甲基化。As_2O_3诱导这些基因启动子区CpG岛去甲基化，并恢复与DNA甲基转移酶抑制相关的表达。As 2 O3还诱导了几个细胞周期相关基因的信使RNA表达的改变，而不依赖于去甲基化。流式细胞仪分析显示As 2 O3诱导的细胞周期阻滞因细胞株而异，MCF-7细胞阻滞于G1期，MDA-MB-231和MDA-MB-468细胞阻滞于G2/M期。这些依赖和不依赖于去甲基化的分子机制导致的细胞周期基因转录水平的变化可能代表了乳腺癌细胞对As 2 O3处理的细胞周期再分配机制。
Arsenic trioxide (As2O3) has been used clinically as an anti-tumor agent. Its mechanisms are mostly considered to be the induction of apoptosis and cell cycle arrest. However, the detailed molecular mechanisms of its anti-cancer action through cell cycle arrest are poorly known. Furthermore, As2O3 has been shown to be a potential DNA methylation inhibitor, inducing DNA hypomethylation. We hypothesize that As2O3 may affect the expression of cell cycle regulatory genes by interfering with DNA methylation patterns. To explore this, we examined promoter methylation status of 24 cell cycle genes in breast cancer cell lines and in a normal breast tissue sample by methylation-specific polymerase chain reaction and/or restriction enzyme-based methods. Gene expression level and cell cycle distribution were quantified by real-time polymerase chain reaction and flow cytometric analyses, respectively. Our methylation analysis indicates that only promoters of RBL1 (p107), RASSF1A, and cyclin D2 were aberrantly methylated in studied breast cancer cell lines. As2O3 induced CpG island demethylation in promoter regions of these genes and restores their expression correlated with DNA methyltransferase inhibition. As2O3 also induced alterations in messenger RNA expression of several cell cycle-related genes independent of demethylation. Flow cytometric analysis revealed that the cell cycle arrest induced by As2O3 varied depending on cell lines, MCF-7 at G1 phase and both MDA-MB-231 and MDA-MB-468 cells at G2/M phase. These changes at transcriptional level of the cell cycle genes by the molecular mechanisms dependent and independent of demethylation are likely to represent the mechanisms of cell cycle redistribution in breast cancer cells, in response to As2O3 treatment.