5-氮杂胞苷可通过抑制Notch1通路诱导食管癌细胞凋亡

背景与目的：5-氮杂胞苷（5-azacytidine，5-azaC）作为一种DNA甲基转移酶抑制剂在临床上已用于多种恶性肿瘤的治疗，但有关5-azaC在食管癌中作用的研究相对较少。凋亡逃逸是肿瘤主要特点之一，也是抗肿瘤治疗研究的热点。Notch1信号通路是促进食管癌发生、发展的重要通路之一，与细胞的增殖、侵袭及凋亡等密切相关。该研究旨在探讨5-azaC对食管癌细胞凋亡的作用及其对Notch1信号通路的影响，从而探索5-azaC作用可能涉及的机制。方法：采用50 μmol/L浓度的5-azaC处理TE-1及OE33两种食管癌细胞系；采用细胞计数试剂盒（cell counting kit-8，CCK-8）检测5-azaC对细胞增殖的抑制效率；采用倒置显微镜观察细胞形态变化；采用流式细胞术、蛋白质印迹法（Western blot）检测两种细胞株在药物处理组与空白对照组的凋亡情况及抗凋亡蛋白B细胞淋巴瘤-XL（B-cell lymphoma-extra large，BCL-XL）的表达；采用实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction，RTFQ-PCR）及Western blot分别检测两种细胞系中Notch1 mRNA表达和蛋白水平的变化及其Notch细胞内区（Notch intracellular domain，NICD）蛋白水平的变化。结果：5-azaC可诱导食管癌细胞TE-1及OE33凋亡，并明显抑制两种细胞增殖；5-azaC作用于TE-1及OE33细胞时可使Notch1 mRNA表达升高，而NICD蛋白表达水平显著下降。结论：5-azaC对食管鳞癌细胞株TE-1及食管腺癌细胞株OE33均有较强的促凋亡作用，其作用机制可能是通过下调NICD蛋白从而抑制Notch1信号通路。

5-azacytidine promotes apoptosis of esophageal cell lines through inhibiting Notch1 pathway

Background and purpose: 5-Azacytidine(5-azaC) is a DNA methyltransferase inhibitor, and has been used to treat many kinds of malignant tumors. However, studies on the role of 5-azaC in esophageal cancer are insufficient. Evading apoptosis is a major feature of tumors and also a hot spot for anti-tumor therapy. Notch1 signaling pathway is one of the most important pathways involved in initiation and development of esophageal cancer, and is closely related to cell proliferation, invasion and apoptosis. This study aimed to investigate the influence of 5-azaC on cell apoptosis and Notch1 signaling pathway in esophageal cell lines, so as to explore the possible mechanism of treatment with 5-azaC. Methods: TE-1 and OE33 cell lines were treated with 5-azaC in a concentration of 50 μmol/L and inhibitory effects of 5-azaC in esophageal cell lines were estimated using cell counting kit-8 (CCK-8). Then cell morphology was visualized using optical microscopy. Flow cytometry and Western blot were used to determine apoptotic rate and to analyze expression of B-cell lymphoma-extra large (BCL-XL), an anti-apoptosis protein, respectively. And finally, realtime fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot were employed to explore expression changesof Notch1 and Notch intracellular domain (NICD). Results: 5-AzaC was able to induce apoptosis, and had a remarkable inhibitory effect on esophageal squamous carcinoma TE-1 and adenocarcinoma cell line OE33. Increased mRNA expression of Notch1 was observed following treatment with 5-azaC, while protein expression of Notch1 was unchanged and NICD showed significant decline in TE-1 and OE33 cell lines. Conclusion: 5-AzaC has strong apoptosis-promoting effect on both esophageal squamous carcinoma TE-1 and adenocarcinoma cell line OE33, and the mechanism may be related to the inhibition of Notch1 signaling pathway via downregulating NICD protein.