Yu Haitao, Zhou Xi, Wang Yanzhong, Huang Xucheng, Yang Jun, Zeng Jin, Li Guoli, Xie Xinyou, Zhang Jun
Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 3 East Qingchun Road, Hangzhou, 310016 Zhejiang People's Republic of China.
Key Laboratory of Biotherapy of Zhejiang Province, 3 East Qingchun Road, Hangzhou, 310016 Zhejiang People's Republic of China.
Cancer Cell Int. 2020 May 24;20:191. doi: 10.1186/s12935-020-01279-8. eCollection 2020.
Nicotinamide N-methyltransferase (NNMT) is highly expressed in several cancers and can regulate cell epigenetic status and various cell metabolism pathways, such as ATP synthesis and cellular stress response. We reported in our previous papers that NNMT overexpression inhibits the apoptosis and enhances the chemotherapy resistance of breast cancer cells. This study aims to investigate the effect of NNMT on autophagy induced by oxidative stress in breast cancer cells, which might provide a novel therapeutic strategy for breast cancer treatment.
NNMT and LC3B II protein levels in the two cell models (SK-BR-3 and MDA-MB-231) with NNMT overexpression or knockdown were detected by Western blotting and correlated with each other. Changes in cellular viability, intracellular reactive oxygen species (ROS) and ATP levels were assessed after HO treatment. Then, autophagosomes were imaged by transmission electron microscopy, and LC3 puncta were examined by confocal microscopy and flow cytometry. The LC3B II level and AMPK-ULK1 pathway activity were both detected by Western blotting to determine the role of NNMT in the HO-induced autophagy.
NNMT expression was negatively correlated with LC3B II expression in both cell models (SK-BR-3 and MDA-MB-231). Then, NNMT overexpression attenuated the autophagy induced by HO in SK-BR-3 cells, whereas knockdown promoted autophagy induced by HO in MDA-MB-231 cells. Furthermore, mechanistic studies showed that NNMT suppressed the ROS increase, ATP decrease and AMPK-ULK1 pathway activation, resulting in the inhibition of HO-induced autophagy in breast cancer cells.
We conclude that NNMT inhibits the autophagy induced by oxidative stress through the ROS-mediated AMPK-ULK1 pathway in breast cancer cells and may protect breast cancer cells against oxidative stress through autophagy suppression.
烟酰胺N-甲基转移酶（NNMT）在多种癌症中高表达，可调节细胞表观遗传状态及各种细胞代谢途径，如ATP合成和细胞应激反应。我们在之前的论文中报道，NNMT过表达可抑制乳腺癌细胞凋亡并增强其化疗耐药性。本研究旨在探讨NNMT对乳腺癌细胞氧化应激诱导的自噬的影响，这可能为乳腺癌治疗提供一种新的治疗策略。
通过蛋白质免疫印迹法检测过表达或敲低NNMT的两种细胞模型（SK-BR-3和MDA-MB-231）中NNMT和LC3B II蛋白水平，并分析二者的相关性。在过氧化氢（HO）处理后评估细胞活力、细胞内活性氧（ROS）水平和ATP水平的变化。然后，通过透射电子显微镜对自噬体进行成像，通过共聚焦显微镜和流式细胞术检测LC3斑点。通过蛋白质免疫印迹法检测LC3B II水平和AMPK-ULK1通路活性，以确定NNMT在HO诱导的自噬中的作用。
在两种细胞模型（SK-BR-3和MDA-MB-231）中，NNMT表达与LC3B II表达均呈负相关。然后，NNMT过表达减弱了HO诱导的SK-BR-3细胞自噬，而敲低则促进了HO诱导的MDA-MB-231细胞自噬。此外，机制研究表明，NNMT抑制了ROS增加、ATP降低和AMPK-ULK1通路激活，从而抑制了乳腺癌细胞中HO诱导的自噬。
我们得出结论，NNMT通过ROS介导的AMPK-ULK1通路抑制乳腺癌细胞氧化应激诱导的自噬，并可能通过抑制自噬保护乳腺癌细胞免受氧化应激。
