RG108 通过抑制 DNMT3B 的表达和 HMLH1 的去甲基化诱导子宫内膜癌细胞系 Ishikawa 凋亡。
RG108 induces the apoptosis of endometrial cancer Ishikawa cell lines by inhibiting the expression of DNMT3B and demethylation of HMLH1.
机构信息
Department of Obstetrics and Gynecology, PLA 463 hospital, Shenyang, China.
出版信息
Eur Rev Med Pharmacol Sci. 2017 Nov;21(22):5056-5064. doi: 10.26355/eurrev_201711_13818.
OBJECTIVE
The effects of DNA methyltransferase (DNMT) inhibitor RG108 on the proliferation and apoptosis of endometrial cancer was investigated, and whether its mechanism was related to the inhibition of DNMT3B, thereby affecting the human mutL homolog 1 (hMLH1) methylation status and its expression, was further studied.
MATERIALS AND METHODS
Culture of human endometrial cancer Ishikawa cell lines: cells grew adhering to the wall in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamic acid). After the cells were treated with RG108, the changes in cell viability were detected via methyl thiazolyl tetrazolium (MTT) assay. The effect of RG108 on cell cycle was detected via flow cytometry, and its effect on cell apoptosis was detected via flow cytometry and TUNEL. Moreover, the methylation status of hMLH1gene in endometrial cancer cells was detected via methylation specific-PCR (MSP), and the changes in DNMT3Band hMLH1 expressions were detected via RT-PCR and Western blotting, respectively.
RESULTS
MTT results showed that RG108 inhibited the cell viability in a dose-dependent and time-dependent manner. Flow cytometry revealed that RG108 blocked the cell cycle in G2/M phase and promoted the apoptosis, and TUNEL assay further proved that RG108 promoted the apoptosis. It was found in the detection via MSP that the methylated hMLH1 gene was significantly reduced after 72 h of treatment with RG108. Besides, RT-PCR and Western blotting showed that RG108 inhibited the DNMT3B expression and activated the hMLH1 expression.
CONCLUSIONS
The demethylation drug RG108 can significantly inhibit the proliferation of endometrial cancer cells, block the cell cycle in the G2/M phase and induce the cell apoptosis, which is a new candidate drug in the treatment of endometrial cancer. RG108 realizes the hMLH1 demethylation and increases the hMLH1 expression through inhibiting the expression of DNMT3B.
目的
研究 DNA 甲基转移酶（DNMT）抑制剂 RG108 对子宫内膜癌细胞增殖和凋亡的影响，并进一步探讨其机制是否与抑制 DNMT3B 从而影响人 mutL 同源物 1（hMLH1）甲基化状态及其表达有关。
材料与方法
人子宫内膜癌细胞系 Ishikawa 的培养：细胞在含有 10%胎牛血清（FBS）和 2 mM L-谷氨酰胺的 Roswell Park Memorial Institute-1640（RPMI-1640）培养基中贴壁生长。用 RG108 处理细胞后，通过甲基噻唑基四唑（MTT）检测细胞活力的变化。通过流式细胞术检测 RG108 对细胞周期的影响，通过流式细胞术和 TUNEL 检测细胞凋亡的影响。此外，通过甲基化特异性 PCR（MSP）检测子宫内膜癌细胞中 hMLH1 基因的甲基化状态，通过 RT-PCR 和 Western blot 分别检测 DNMT3B 和 hMLH1 表达的变化。
结果
MTT 结果表明，RG108 呈剂量和时间依赖性抑制细胞活力。流式细胞术显示，RG108 阻滞细胞周期于 G2/M 期并促进细胞凋亡，TUNEL 检测进一步证实 RG108 促进细胞凋亡。通过 MSP 检测发现，用 RG108 处理 72 小时后，甲基化 hMLH1 基因明显减少。此外，RT-PCR 和 Western blot 显示，RG108 抑制 DNMT3B 表达并激活 hMLH1 表达。
结论
去甲基化药物 RG108 可显著抑制子宫内膜癌细胞增殖，阻滞细胞周期于 G2/M 期并诱导细胞凋亡，是治疗子宫内膜癌的一种新候选药物。RG108 通过抑制 DNMT3B 的表达实现 hMLH1 的去甲基化并增加 hMLH1 的表达。
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