zebularine调控卵巢癌A2780细胞RASSF1A基因再表达的研究

目的:通过观察应用去甲基化药物zebularine(Zeb)后人卵巢癌细胞系A2780中抑癌基因RASSF1A甲基化状态及表达水平的改变,探讨 Zeb对卵巢癌细胞RASSF1A基因表达的调控.方法:应用不同浓度(50、100、200 μmol·L-1) 的Zeb处理体外培养的卵巢癌细胞A2780后,甲基化特异性PCR(...     

The DNA methyltransferase inhibitors zebularine and decitabine induce mitochondria-mediated apoptosis and DNA damage in p53 mutant leukemic T cells

DNA methyltransferase (DNMT)-inhibiting nucleoside analogs reactivate the expression of tumor suppressor genes and apoptosis-related genes silenced by methylation, thus favoring the induction of apoptosis in tumor cells. Moreover, induction of DNA damage seems to contribute to their antitumor effect. However, the apoptotic signaling pathway induced by these demethylating drugs is not well understood. Here, we have investigated the induction of apoptosis by two nucleoside DNMT inhibitors, decitabine and zebularine, in leukemic T cells. Both inhibitors induced caspase-dependent apoptosis in Jurkat, CEM-6 and MOLT-4 leukemia T cell lines, all with mutant p53, whereas resting and activated normal T lymphocytes were highly resistant to these demethylating agents. Although decitabine and zebularine showed different ability to induce apoptosis and cell cycle arrest among the three cell lines, they similarly activated the intrinsic apoptotic pathway by inducing mitochondrial alterations such as Bak activation, loss of transmembrane potential and generation of reactive oxygen species (ROS). Accordingly, Bcl-2- and Bcl-x(L) -overexpressing Jurkat cells, as well as caspase-9-deficient Jurkat cells, were resistant to apoptosis induced by decitabine and zebularine. Interestingly, ROS production seemed to be necessary for the induction of apoptosis. Apoptotic events, such as Bak and caspase activation, started as soon as 20 hr after treatment with either decitabine or zebularine. In addition, progression of apoptosis triggered by both DNMT inhibitors was paralleled by the induction of DNA damage. Our results suggest that the mitochondrial apoptotic pathway activated by decitabine and zebularine in p53 mutant leukemic T cells depends mainly on the induction of DNA damage.     

Zebularine elevates STING expression and enhances cGAMP cancer immunotherapy in mice

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Zebularine 调控卵巢癌细胞hMLH1基因再表达

 目的探讨去甲基化药物Zebularine（Zeb）对体外培养的顺铂耐药的卵巢癌细胞A2780/cp70 hMLH1基因甲基化状态的
影响及表达调控作用。方法应用不同浓度的Zeb（50 μmol/L,100 μmol/L,200 μmol/L）处理体外培养的卵巢癌细胞A2780/
cp70 后,甲基化特异性PCR（Methylation-specific PCR,MSP）法检测用药前后细胞中hMLH1基因的甲基化状态,RT-PCR 法及
Western-blot 法检测用药前后细胞中hMLH1基因mRNA及蛋白表达的变化。结果hMLH1基因在卵巢癌细胞A2780/cp70 中
呈异常甲基化状态,在mRNA及蛋白水平表达阴性。经过Zeb 处理后,hMLH1基因呈去甲基化状态,其mRNA及蛋白重新表
达。结论异常甲基化是卵巢癌细胞A2780/cp70 hMLH1基因失活的原因,去甲基化药物Zeb 能使hMLH1基因去甲基化从而
调控其重新表达。     

zebularine对卵巢癌细胞的生长抑制作用

目的:观察去甲基化制剂zebularine(Zeb)对体外培养的卵巢癌细胞A2780的生长抑制作用,并进一步探讨其作用机制。方法:应用不同浓度(50、100、200μmo.lL-1)的Zeb处理体外培养的卵巢癌细胞A2780;应用MTT法检测细胞增殖活性,流式细胞仪检测细胞周期分布,Annexin V-FITC染色法检测细胞凋亡率。结果:100、200μmo.lL-1的Zeb处理卵巢癌细胞A2780 24~72 h后,细胞增殖显著受到抑制(P<0.05);100、200μmo.lL-1的Zeb处理72 h后A2780细胞G2期细胞数量显著增加(P<0.05),S期细胞数显著减少(P<0.05);100、200μmo.lL-1组细胞凋亡率均增加(P<0.05)。结论:Zeb对卵巢癌细胞A2780具有生长抑制作用,其机制与阻滞细胞周期及诱导凋亡有关。     

DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.